Mean of strain

**the strains** 

(25)

(10)

(5)

Figure in parenthesis indicate percent over sensitive strain

Agriculture crops are under continuous attack of serious and noxious organisms. They are powerful challengers of nature and manmade technologies. To safeguard world food production, crop protection measures especially choice based intelligent and ecofriendly chemicals are indispensable as they are instant and with known mode of action.

However, control of pest and disease with chemicals also encounter several problems, when the pathogens resistance to the potential broad-spectrum chemical. The ability of a pathogen to develop stable resistance to any toxicant is the fundamental theory of survival of the fittest under unfavorable conditions. The evolution of organisms would have been impossible without this property.

In a fungal population that is originally sensitive to fungicide, forms may arise or already exist that are less sensitive to the fungicide. Such a decrease in sensitivity may be caused by genetic or non-genetic changes in the fungal cell. Decrease in sensitivity due to genetic changes in the pathogen is more serious and requires in-depth understanding to detect the resistance in the field and combat the resistance well in advance to avoid or delay build of resistance.

Progress in clarifying the biochemical mechanisms of resistance has been made with some systemic fungicides, viz. Carbendazim, Carboxamides and Organophosphorous compounds (Georgopoulos, 1977). However, it is usually better to act before the buildup of resistance starts. For this, one should collect information from experiments with test fungi in vitro about the chances of a resistance problem arising in practice. Laboratory mutants resistant to test fungicides can be developed through in vitro mutagenesis (mutagenic chemicals or Ultra violet irradiation). The wild or parent strains should be characterized throughout to compare the resistant mutants. Also, the wild strain should be thoroughly characterized for its sensitivity against the target fungicide. In the present studies paddy pathogen *B. oryzae* was taken as test pathogen.

Detailed chemical control work has recommended benzimidazoles as one of the best broad spectrum compound for the control of paddy pathogen, *B. oryzae*. Present study is an attempt to predict the disease control failure using modern molecular diagnostic method to detect under in vitro condition to combat the development of resistance.

Before proceeding to produce Carbendazim resistant strains the sensitivity of the test pathogens was carefully monitored and ED 50 value of carbendazim for all test pathogens *B. oryzae*. Laboratory mutants were developed through UV irradiation, EMS mutagenesis and adaptation. There are several reports on the development of resistant mutants through EMS, UV irradiation and adaptation (Sanchez et al., 1975, Davidse, 1981, Leach et al., 1982; Gangawane et al., 1988; Rana and Sengupta, 1977, Hillderbrand et al., 1988). Many of the laboratory mutants were stable with high level of resistance.

In the present studies, though resistance to Carbendazim in test pathogens was suspected to be site modification, it was made very clear through results that site modifications is quiet rare and is not frequently observed in fields under practice with target fungicides.

PCR amplification of β-tubulin and spot hybridization further confirmed that the resistance at various levels tested proved that there was no point mutation observed in all the test pathogens. Instead ample evidences are present to confirm that in majority of the test pathogens, the mechanism of resistance primarily and always observed in membrane modification which could easily be handled or controlled easily with negatively correlated chemicals.

Hence, the present studies had given an authentic molecular proof that all benzimodazole resistance observed need not be site modifications which is very difficult to manage but mostly membrane modification, clearly indicating the possibility that fungicide resistance at any level if predicted can easily be managed and it is recommended that selection of choice based chemical (alternate chemical) with careful monitoring will definitely give a stable and sustainable management of diseases successfully in the agricultural practices. This is an additional warranty for the management of uncontrollable diseases using biological control.

#### **5. Summary**

Many pathogens develop resistance under field conditions due to frequent application of fungicides. To evaluate the resistance risk, stable laboratory mutant of *B. oryzae* resistant to Carbendazim were developed under laboratory conditions. Important pathogen *B. oryzae* was selected for intensive studies on molecular mechanisms of the fungicide resistance to benzimidazole compound. For the precise understanding of resistant mutants the complete characterization of parent strains very carefully carried out. The growth kinetics of *B. oryzae* was estimated on PDA. Similarly the dry weights of the test pathogen were estimated under static and shaken conditions. The mycelial dry weight was more under shaken condition than the static condition. Test pathogens were screened for sensitivity against Carbendazim using different concentration. The ED50 value for *B. oryzae* was 40 M.

Induction of laboratory mutants using EMS, UV irradiation and adaptation technique were carried out. More than 200 resistant colonies were taken to select resistant strains 200 colonies were screened on PDA amended with 5 times the ED50 value of Carbendazim. The respective sensitive strains and resistant colonies were picked up and stored in test tube slants amended with test fungicides. The percent survival was assessed for all the test pathogens. Stability of resistance in all the mutants were checked. All the stable mutants of test pathogens when tested for pathogenicity proved to be pathogenic by causing respective disease through artificial inoculation. All the stable resistant mutants were categorized as LR, MR, HR and VHR based on their level of resistance over ED50 values of sensitive strain. Selected mutants when grow on (5 – 10 times LR, 10 –15 times MR, 15-20 times HR and 20 – 25 times VHR). Sixty mutants in each pathogen were screened on the above concentration of Carbendazim and categorized as LR, MR, HR and VHR. Sporulation of the resistant mutants were evaluated for all test pathogens and compared. In sensitive strains more sporulation was observed than the sensitive strains. Agarose gel electrophoresis of genomic DNA of the all the levels of mutants of test pathogens did not show any variation. Cross resistance study showed that *B. oryzae* resistant mutants at all levels could be overcome by alternative use of Dithane-45 or Mancozeb. The PCR amplification of β-tubulin in DNA extract was very poor and in most of the resistant mutants DNA, β–tubulin could not be amplified.

#### **6. Acknowledgment**

Authors have to thanks to University of Guyana, Berbice Campus to financially support this book chapter and also special thanks to Prof. Daizal R.Samad, Director, Berbice Campus to fully support us to write a book chapter.

#### **7. References**

216 Fungicides – Beneficial and Harmful Aspects

Progress in clarifying the biochemical mechanisms of resistance has been made with some systemic fungicides, viz. Carbendazim, Carboxamides and Organophosphorous compounds (Georgopoulos, 1977). However, it is usually better to act before the buildup of resistance starts. For this, one should collect information from experiments with test fungi in vitro about the chances of a resistance problem arising in practice. Laboratory mutants resistant to test fungicides can be developed through in vitro mutagenesis (mutagenic chemicals or Ultra violet irradiation). The wild or parent strains should be characterized throughout to compare the resistant mutants. Also, the wild strain should be thoroughly characterized for its sensitivity against the target fungicide. In the present studies paddy pathogen *B. oryzae*

Detailed chemical control work has recommended benzimidazoles as one of the best broad spectrum compound for the control of paddy pathogen, *B. oryzae*. Present study is an attempt to predict the disease control failure using modern molecular diagnostic method to

Before proceeding to produce Carbendazim resistant strains the sensitivity of the test pathogens was carefully monitored and ED 50 value of carbendazim for all test pathogens *B. oryzae*. Laboratory mutants were developed through UV irradiation, EMS mutagenesis and adaptation. There are several reports on the development of resistant mutants through EMS, UV irradiation and adaptation (Sanchez et al., 1975, Davidse, 1981, Leach et al., 1982; Gangawane et al., 1988; Rana and Sengupta, 1977, Hillderbrand et al., 1988). Many of the

In the present studies, though resistance to Carbendazim in test pathogens was suspected to be site modification, it was made very clear through results that site modifications is quiet

PCR amplification of β-tubulin and spot hybridization further confirmed that the resistance at various levels tested proved that there was no point mutation observed in all the test pathogens. Instead ample evidences are present to confirm that in majority of the test pathogens, the mechanism of resistance primarily and always observed in membrane modification which could easily be handled or controlled easily with negatively correlated

Hence, the present studies had given an authentic molecular proof that all benzimodazole resistance observed need not be site modifications which is very difficult to manage but mostly membrane modification, clearly indicating the possibility that fungicide resistance at any level if predicted can easily be managed and it is recommended that selection of choice based chemical (alternate chemical) with careful monitoring will definitely give a stable and sustainable management of diseases successfully in the agricultural practices. This is an additional warranty for the management of uncontrollable diseases using biological control.

Many pathogens develop resistance under field conditions due to frequent application of fungicides. To evaluate the resistance risk, stable laboratory mutant of *B. oryzae* resistant to Carbendazim were developed under laboratory conditions. Important pathogen *B. oryzae* was selected for intensive studies on molecular mechanisms of the fungicide resistance to

rare and is not frequently observed in fields under practice with target fungicides.

detect under in vitro condition to combat the development of resistance.

laboratory mutants were stable with high level of resistance.

was taken as test pathogen.

chemicals.

**5. Summary** 


### **Accuracy of Real-Time PCR to Study**  *Mycosphaerella graminicola* **Epidemic in Wheat: From Spore Arrival to Fungicide Efficiency**

Selim Sameh, Roisin-Fichter Céline, Andry Jean-Baptiste and Bogdanow Boris *Platform Biotechnology and Plant Pathology, Institut Polytechnique LaSalle Beauvais, GIS PhyNoPi: Groupement d'Intérêt Scientifique «Phytopathologie Nord-Picardie», France* 

#### **1. Introduction**

218 Fungicides – Beneficial and Harmful Aspects

Johnson M.J.1972. Continuous measurment of dissolved oxygen. In; Manometric and

Laemmii.U.K.1970. Clevage of structural protein using the assembly of the head of

Lalithakumari,.D, J.R.Decallonne and J. A. Meyer. 1975. M.Talpaert.1977. Interferance of

nuclear divisions in Fusarium oxysporum, *Pestic.Biochem.Physiol*.7:273-282. Leech, J., Lang, B.R., and Yoder B.C. 1982. Methods for selection of mutants and in votro culture of *Cochliobolus heterostrophus*. *J. Gen. Microbiol*. 128: 1719 – 1729. Munro, H.N. and A.Fleck.1966. The determination of nucleic acids. In: Netherlands

Rana;J.P. and P.K. Sengupta. 1977. Adaptationof two plants pathogenic fungi to some

Sanchez, L.E., J.V. Leary and R.M. Endo. 1975.Chemical mutagenesis of Fusarium

Tanaka,C.,Y.Kubo and M.Tsuda.1988.Comparison of mutagens in *Cochliobo heterostrophus*

132. Burgess Pubishing Co. Minnesota.

bactriaphages. Nature (London).680-685.

mutants. *J. Gen. Microbiol*. 87:326-332.

fungicide. *Z. Pflanzenkrankh. Pflanzensch*. 84:738-742

mutagenesis. Ann.Phytopathol.Soc.Jpn.54: 503-509. Welch, P.S. 1948. *Limnological methods*. P. 306. McGraw Hill, Inc.

biochemical techniques (Eds) W.W.Umbrieit R.H.Burris and J.F.Stauffer.pp.126-

bnomyl and methyl-2-benzimidazole carbamate (MBC) with DNA metabolism and

Biochemical analysis (Ed.) D. Glick. p.113-176. Inter Science Publishers, New York.

oxysporum f.sp. lycopersici: Non-selected changes in pathogenicity of auxotrophic

*Mycosphaerella graminicola* (Fuckel) J. Schroeter in Cohn (anamorph *Septoria tritici* Roberge in Desmaz.) is one of the most important pathogens on winter wheat in northern Europe (Leroux et al., 2007; Stammler et al., 2008b). This fungus is responsible for *Septoria* leaf blotch and causes 3040% yield reduction under extreme conditions (Palmer & Skinner, 2002). The primary inoculum is commonly described by the arrival of *M. graminicola* ascospores (Shaw & Royle, 1989). At high humidity, spores germinate and germ tubes penetrate 12 hours post inoculation into the leaves, exclusively through the stomata. The internal colonization is still intercellular between mesophyll cells until 1012 days post inoculation. Then, the host cells die in response to the necrotrophic mode of *S. tritici,* with the development of visual chlorosis and necrotic symptoms. The formation of brown/black pycnidia in the substomatal cavities of the necrotic spots results from asexual reproduction, which appears 1421 days after inoculation (Kema et al., 1996). Pycnidiospores are the secondary inoculum and are responsible for the repetition of the infection in the upper foliar layers by the rain effect on the vertical spore transfer, called "splashing" (Shaw & Royle, 1993).

The application of fungicides is the most common method used for controlling this pathogen. Several families of fungicides exist and are used against *M. graminicola*, but their efficiencies differ and decline with use (Fraaije & Cools, 2008). The azole class of sterol demethylation inhibitors (DMIs), which includes triazoles, is able to control *S. tritici* targeting of the *CYP51* enzyme (14-demethylase), and it has been used frequently over the past 25 years (Leroux et al., 2007; Stammler et al., 2008b). Over the past 20 years, however, significant changes in the sensitivity of *M. graminicola* strains toward DMI fungicides have been widely reported (Leroux et al., 2007), especially over the past five years in the case of triazoles.

Resistance to DMIs has been described by various mechanisms, one of which is point mutations in the *CYP51* gene, which reduces the affinity between DMIs and the 14 demethylase enzyme (Leroux et al., 2007; Stammler et al., 2008b). To date, 17 DMI-resistant genotypes (R-types) characterized by amino acid alterations have been reported in the *M. graminicola CYP51* gene: L50S, V136A/C, A379G, S188N (Leroux et al., 2007); G510C, N513K, Y137F (Cools et al., 2005; Leroux et al., 2007); I381V (Cools et al., 2005; Leroux et al., 2007); and Y459D/S/N/C, G460D, Y461H/S, Y459/G460 (Cools et al., 2005; Leroux et al., 2007). Each *CYP51* variant is selected differently by triazole fungicides, and there are two main categories: strains that contain the isoleucine amino acid at the position 381 of the *CYP51* protein sequence (I381) and strains with the point mutation (V381), where isoleucine is replaced by valine (Leroux et al., 2007; Stammler et al., 2008b).

As this disease has a long latent period, presymptomatic detection and quantification of *M. graminicola* and genotypes that are related to different DMI resistance levels are very important for the effective control of strains that are highly resistant to DMIs. This allows better timing, choice and dose of fungicide applications. However, it is not possible to achieve such detection by conventional methods, and hence fungicides cannot be applied until visible symptoms appear.

Molecular detection of disease in plants was developed using the polymerase chain reaction (PCR) technique, which offers a rapid and sensitive diagnostic method (Henson and French 1993). The association of specific chemistries and a fluorescent reporter molecule (TaqMan chemistry) with PCR has permitted the development of real-time PCR (Schena et al., 2004). This technique is an accurate and reliable tool for many phytopathological studies (Guo et al., 2006 & 2007; Fraaije et al., 1999; Schena et al., 2004). Real-time PCR analysis, also referred to as quantitative PCR (qPCR), can provide a deeper insight into host–pathogen interactions by detecting the primary inoculum, general infection level and genotypes, depending on the specific target gene. For *M. graminicola*, qPCR based on the specific and stable gene -tubulin has been described as a diagnostic tool by (Bearchell et al., 2005) and on the *CYP51* gene for 14-demethylase mutations by (Selim, 2009).

In the present study, we used qPCR to study the epidemiological context of *M. graminicola,* taking into account the effect of many factors, including external contamination by ascospores, cultivars resistance, leaf colonization stages and fungicide efficiency. The correlation between results of molecular qPCR analysis and visual symptoms observations were investigated.

#### **2. Materials and methods**

#### **2.1 In vivo studies of** *Septoria* **leaf blotch using qPCR**

#### **2.1.1 Trapping of spores**

The spore trap assay is described by (Fraaije et al., 2005). A spore trap was installed in the field, at a distance of 3 m from the wheat trial in order to avoid the capture of pycnidiospores that move vertically as a result of splashing raindrops. Spores were collected from a plastic film, and then they were ground using an MM 300 Mixer Mill (Qiagen, USA) in a 2-ml Eppendorf Safe-lock micro test tube containing 250 mg white quartz (Sigma S-9887) and one 5-mm stainless steel bead (Cat. No. 69989, Qiagen, France).

#### **2.1.2 Evolution of** *Septoria* **leaf blotch and resistant wheat cultivars**

The field sites were located at the Beauvais Agricultural Research Station of the Institut Polytechnique LaSalle-Beauvais, Beauvais, France. Based on the susceptibility to *M. graminicola*, one resistant cultivar (Maxwell) and four susceptible cultivars (Dinosor, Alixan, Trémie and Maxyl) were selected (Table 1). Rainfall, temperature and leaf wetness in the trial field were monitored daily (Figure 1a). Trials were performed during the 2008–2009 growing season using a completely randomized block design with three replicate plots. Fertilization was according to plant requirements and protection against other diseases was carried out. The plot size was 2 12 m. Twenty leaves were randomly sampled from one foliar layer of the main stem. The foliar layer number (Fn) was determined by counting the position of the leaf from the flag leaf (F1). Disease evolution was determined by assessing visual symptoms and by qPCR analysis. The necrotic area related to Septoria blotch was recorded and then the leaves were stored at 80 C until lyophilization.


\* Susceptibility rating (19): 1 (susceptible) to 9 (resistant).

Table 1. Wheat cultivars used in the resistance study against *Mycosphaerella graminicola*

#### **2.1.3** *S. tritici* **DMI-resistant genotypes**

220 Fungicides – Beneficial and Harmful Aspects

and Y459D/S/N/C, G460D, Y461H/S, Y459/G460 (Cools et al., 2005; Leroux et al., 2007). Each *CYP51* variant is selected differently by triazole fungicides, and there are two main categories: strains that contain the isoleucine amino acid at the position 381 of the *CYP51* protein sequence (I381) and strains with the point mutation (V381), where isoleucine is

As this disease has a long latent period, presymptomatic detection and quantification of *M. graminicola* and genotypes that are related to different DMI resistance levels are very important for the effective control of strains that are highly resistant to DMIs. This allows better timing, choice and dose of fungicide applications. However, it is not possible to achieve such detection by conventional methods, and hence fungicides cannot be applied

Molecular detection of disease in plants was developed using the polymerase chain reaction (PCR) technique, which offers a rapid and sensitive diagnostic method (Henson and French 1993). The association of specific chemistries and a fluorescent reporter molecule (TaqMan chemistry) with PCR has permitted the development of real-time PCR (Schena et al., 2004). This technique is an accurate and reliable tool for many phytopathological studies (Guo et al., 2006 & 2007; Fraaije et al., 1999; Schena et al., 2004). Real-time PCR analysis, also referred to as quantitative PCR (qPCR), can provide a deeper insight into host–pathogen interactions by detecting the primary inoculum, general infection level and genotypes, depending on the specific target gene. For *M. graminicola*, qPCR based on the specific and stable gene -tubulin has been described as a diagnostic tool by (Bearchell et al., 2005) and on the *CYP51* gene for

In the present study, we used qPCR to study the epidemiological context of *M. graminicola,* taking into account the effect of many factors, including external contamination by ascospores, cultivars resistance, leaf colonization stages and fungicide efficiency. The correlation between results of molecular qPCR analysis and visual symptoms observations were investigated.

The spore trap assay is described by (Fraaije et al., 2005). A spore trap was installed in the field, at a distance of 3 m from the wheat trial in order to avoid the capture of pycnidiospores that move vertically as a result of splashing raindrops. Spores were collected from a plastic film, and then they were ground using an MM 300 Mixer Mill (Qiagen, USA) in a 2-ml Eppendorf Safe-lock micro test tube containing 250 mg white quartz (Sigma S-

The field sites were located at the Beauvais Agricultural Research Station of the Institut Polytechnique LaSalle-Beauvais, Beauvais, France. Based on the susceptibility to *M. graminicola*, one resistant cultivar (Maxwell) and four susceptible cultivars (Dinosor, Alixan, Trémie and Maxyl) were selected (Table 1). Rainfall, temperature and leaf wetness in the trial field were monitored daily (Figure 1a). Trials were performed during the 2008–2009 growing season using a completely randomized block design with three replicate plots.

replaced by valine (Leroux et al., 2007; Stammler et al., 2008b).

until visible symptoms appear.

**2. Materials and methods** 

**2.1.1 Trapping of spores** 

14-demethylase mutations by (Selim, 2009).

**2.1 In vivo studies of** *Septoria* **leaf blotch using qPCR** 

9887) and one 5-mm stainless steel bead (Cat. No. 69989, Qiagen, France).

**2.1.2 Evolution of** *Septoria* **leaf blotch and resistant wheat cultivars** 

*S. tritici* DMI-resistant genotypes were determined for wheat leaf samples collected from the five wheat cultivars (listed above) grown under field conditions. Intentional mismatch primers (Table 2) and allele-specific qPCR were used as described by (Selim, 2009), to quantify the mutation proportions of *CYP51* in DNA samples.


Lower case nucleotide indicates the intentional mismatch, nucleotides in bold are located at the site of single nucleotide polymorphism (SNP), (for) and (rev) indicate forward and reverse orientation, (W) indicates wild-type specific primer, (Tm) indicates primer melt temperature, (//) and **Y**459/**G**460 indicate double deletion of tyrosine (Y) and glycine (G) at positions 459 and 460 of the amino acid sequence, respectively.

Table 2. Sequences of primers and probes used to determine moderate resistant (R6, R7 or R7+) or general (MG) genotypes of *M. graminicola*

#### **2.2** *In vitro* **studies of** *Septoria* **leaf blotch using qPCR**

#### **2.2.1 Plant material**

Susceptible and resistant winter wheat cultivars Dinosor and Maxwell, respectively, were used. Grains were disinfected by incubation with 1% sodium hypochlorite for 5 minutes, with shaking, and then washed three times in autoclaved distilled water. Grain germination was realized in a 0.5% water–agar medium. After incubation in darkness; 24 hours at 20 C, 48 hours at 4 C and then 24 hours at 20 C (Arraiano et al. 2001), grains were transferred into 500 ml pots containing an autoclaved soil mixture of horticultural compost, sand and silt–loam soil (1:1:2 v/v/v). Pots were incubated at 15 C, for a 16-hour photoperiod, with 150 mol of photon.m–2.s1. Plantlets were watered twice a week with 50 ml distilled water per pot, and once a week with 50 ml water containing 25% Murashige and Skoog basal medium (Sigma M 5519).

#### **2.2.2 Inoculum preparation**

*M. graminicola* isolates T0248, T0254 and T0256 were obtained from *S. tritici* collection strains held in the authors' laboratory. Sporidia (yeast-like cells) stored at 80 C were activated by transfer to fresh potato dextrose agar medium (39 g.l–1, Sigma, USA). After 10 days of incubation at 18 C, with a 12-hour photoperiod, mycelia and spores were scraped off the surface and grown in a liquid yeast–sucrose medium (yeast extract 10 g.l–1, sucrose 10 g.l–1; Sigma, USA) for 7 days at 18 C with permanent light (100 mol of photon.m–2.s1) and shaking (150 rpm). Spores were collected by centrifugation at 1500 rpm for 5 minutes at 15 C, washed twice with sterile distilled water, and then suspended in 10 mM MgSO4 (Sigma M-9397) containing 0.1% Tween 20 surfactant. The concentration was adjusted to 105 spores.ml1.

#### **2.2.3 Plantlet inoculation and leaf sampling**

Twenty-one-day-old plantlets were inoculated either with 10 l (one drop) of *M. graminicola*  inoculum (105 spores.ml1) at the bottom part of the second leaf for microscopic observations, or by spraying a 3 ml covering over the whole plantlet to assess fungicide efficiency. The plants were enveloped in a transparent polyethylene bag for 3 days. The bag provided an atmosphere of saturated humidity around the inoculated leaves. Disease development was followed for 1 month after inoculation by sampling inoculated leaves at 0, 1, 2, 3, 5, 7, 12, 14, 16, 19, 23, 26, 28 and 30 days post inoculation (dpi). Six leaves per leaf layer were collected each time, three for the microscopic observations and three for qPCR analyses.

#### **2.2.4 Fungicide treatments**

JOAO (prothioconazole 250 g a.i. l–1, Bayer CropSciences, Langenfeld, Germany) in its recommended dose (0.8 l.ha1) was tested for its efficiency in containing the development of *Septoria* disease under controlled conditions. Depending on the day of inoculation (d0), four modalities of fungicide application were tested: one preventive modality at day one before inoculation (d1) and three curative modalities at 3, 7 and 10 days after inoculation (d+3, d+7 and d+10, respectively). Each plant was covered once with 3 ml of the fungicide solution, supplemented with one drop of Tween 20, using a TLC sprayer (Grace, USA).

#### **2.2.5 Microscopic observations**

222 Fungicides – Beneficial and Harmful Aspects

Susceptible and resistant winter wheat cultivars Dinosor and Maxwell, respectively, were used. Grains were disinfected by incubation with 1% sodium hypochlorite for 5 minutes, with shaking, and then washed three times in autoclaved distilled water. Grain germination was realized in a 0.5% water–agar medium. After incubation in darkness; 24 hours at 20 C, 48 hours at 4 C and then 24 hours at 20 C (Arraiano et al. 2001), grains were transferred into 500 ml pots containing an autoclaved soil mixture of horticultural compost, sand and silt–loam soil (1:1:2 v/v/v). Pots were incubated at 15 C, for a 16-hour photoperiod, with 150 mol of photon.m–2.s1. Plantlets were watered twice a week with 50 ml distilled water per pot, and once a week with 50 ml water containing 25% Murashige and Skoog basal

*M. graminicola* isolates T0248, T0254 and T0256 were obtained from *S. tritici* collection strains held in the authors' laboratory. Sporidia (yeast-like cells) stored at 80 C were activated by transfer to fresh potato dextrose agar medium (39 g.l–1, Sigma, USA). After 10 days of incubation at 18 C, with a 12-hour photoperiod, mycelia and spores were scraped off the surface and grown in a liquid yeast–sucrose medium (yeast extract 10 g.l–1, sucrose 10 g.l–1; Sigma, USA) for 7 days at 18 C with permanent light (100 mol of photon.m–2.s1) and shaking (150 rpm). Spores were collected by centrifugation at 1500 rpm for 5 minutes at 15 C, washed twice with sterile distilled water, and then suspended in 10 mM MgSO4 (Sigma M-9397) containing 0.1% Tween 20 surfactant. The concentration was adjusted to 105

Twenty-one-day-old plantlets were inoculated either with 10 l (one drop) of *M. graminicola*  inoculum (105 spores.ml1) at the bottom part of the second leaf for microscopic observations, or by spraying a 3 ml covering over the whole plantlet to assess fungicide efficiency. The plants were enveloped in a transparent polyethylene bag for 3 days. The bag provided an atmosphere of saturated humidity around the inoculated leaves. Disease development was followed for 1 month after inoculation by sampling inoculated leaves at 0, 1, 2, 3, 5, 7, 12, 14, 16, 19, 23, 26, 28 and 30 days post inoculation (dpi). Six leaves per leaf layer were collected each time, three for the microscopic observations and three for qPCR

JOAO (prothioconazole 250 g a.i. l–1, Bayer CropSciences, Langenfeld, Germany) in its recommended dose (0.8 l.ha1) was tested for its efficiency in containing the development of *Septoria* disease under controlled conditions. Depending on the day of inoculation (d0), four modalities of fungicide application were tested: one preventive modality at day one before inoculation (d1) and three curative modalities at 3, 7 and 10 days after inoculation (d+3,

**2.2** *In vitro* **studies of** *Septoria* **leaf blotch using qPCR** 

**2.2.1 Plant material** 

medium (Sigma M 5519).

**2.2.2 Inoculum preparation** 

**2.2.3 Plantlet inoculation and leaf sampling** 

spores.ml1.

analyses.

**2.2.4 Fungicide treatments** 

Microscopic observations were carried out according to (Shetty et al., 2003), with modifications. Leaves were cut and cleared by placing them overnight between two filter papers saturated with glacial acetic acid and absolute ethanol (1:3 v/v). They were then washed three times with distilled water and stored between two filter papers in a solution of lactoglycerol (lactic acid/glycerol/distilled water, 1:1:1, v/v/v) until observation. Coloration was carried out by incubating the leaf parts in 0.1% trypan blue in lactoglycerol for 1 hour at 50 C.

Stained slides were microscopically assessed using a Leica DM 4500P research microscope (Leica Microsystems, Bensheim, Germany). Further magnification was achieved by analyzing the surface of cryofractured nonstained leaf fragments with an electron microscope (TM-1000 Tabletop Microscope; HITACHI High-Technology Corporation, Japan).

#### **2.2.6 DNA extraction**

All plant leaves analyzed by qPCR had less than 40% necrotic surface. Collected leaf samples were placed directly in liquid nitrogen and then lyophilized in a Virtis 12 XL lyophilizer for 48 hours. The dried samples were then ground using an MM 300 Mixer Mill (Qiagen, USA). DNA was extracted using the DNeasy 96 Plant kit (Qiagen, USA) according to the manufacturer's protocol. DNA was quantified by measurement of UV absorption at 260 nm (Cary 50 UV–Vis spectrophotometer; Varian, France).

#### **2.2.7 Quantification of** *S. tritici* **using qPCR analysis**

To quantify infection levels of *S. tritici*, primers and TaqMan minor groove binder probes were used to target a 63-bp fragment of the -tubulin gene (GenBank accession no. AY547264), as described in (Bearchell et al., 2005). A TaqMan assay was carried out in 25 l of a reaction mixture that contained the following: 12.5 l Universal TaqMan PCR Master Mix (Applied Biosystems, USA), 0.3 M of each primer, 0.2 M probe, 200 ng DNA and water up to a volume of 25 l. The conditions of qPCR determination were the following: 10 minutes at 95 C, followed by 40 cycles of 15 seconds at 95 C and 1 minute at 60 C. All qPCR experiments were carried out using an ABI PRISM 7300 sequence detection system (Applied Biosystems, USA).

qPCR analysis of the *M. graminicola* -tubulin gene was calibrated from 102 to 107 copies by serial dilution of the appropriate cloned target sequence.

#### **3. Results**

#### **3.1 External contamination by ascospores**

Data in figure 1a show that there was dry weather during the experimental season (2008– 2009) and low temperatures of around 10 C until the end of April. Results of spore capture showed a low level of external ascospore contamination (Figure 1b). Three periods of contamination were observed. The first period was during April, with two main peaks that represented 30 and 3000 ascospores per day. The second was the main period, which represented a continuous arrival of ascospores during May, with a stable number of about 100 spores per day. The third period was a classical period of ascospore production, which was at the end of the wheat growing season during July (about 30 spores per day).

Fig. 1. (a) Weather conditions and (b) temporal dispersal of ascospores in *M. graminicola* DNA presented as the amount per day.

#### **3.2 Evolution of fungal biomass**

Evolution of *S. tritici* was characterized by simultaneously using visual symptoms and *M. graminicola* -tubulin gene qPCR (Figure 2). Observation of necrotic areas on the top three leaf layers of the two cultivars Maxwell and Dinosor showed late disease development

capture showed a low level of external ascospore contamination (Figure 1b). Three periods of contamination were observed. The first period was during April, with two main peaks that represented 30 and 3000 ascospores per day. The second was the main period, which represented a continuous arrival of ascospores during May, with a stable number of about 100 spores per day. The third period was a classical period of ascospore production, which was at the end of the wheat growing season during July (about 30

Fig. 1. (a) Weather conditions and (b) temporal dispersal of ascospores in *M. graminicola*

Evolution of *S. tritici* was characterized by simultaneously using visual symptoms and *M. graminicola* -tubulin gene qPCR (Figure 2). Observation of necrotic areas on the top three leaf layers of the two cultivars Maxwell and Dinosor showed late disease development

DNA presented as the amount per day.

**3.2 Evolution of fungal biomass** 

spores per day).

during June, with a "croissant"-like gradient from the top of the plant (flag leaf) to the bottom. The epidemic began in the susceptible cultivar Dinosor 15 days earlier than in the resistant cultivar Maxwell. By 16 June (Zadok's growth stage (GS) 85), Dinosor's leaf necrotic surface was 5, 22 and 25% for F1, F2 and F3, respectively, whereas Maxwell had not shown any symptoms. Two weeks later, all three leaf layers of Dinosor had more than 60% necrotic surface, whereas the values for Maxwell were 11.6, 23.5 and 67.5%, respectively.

Fig. 2. Septoria blotch disease progression on Dinosor and Maxwell cultivars measured by necrotic surface observation and by qPCR.

The development of epidemics of *M. graminicola* was detected earlier with qPCR analysis (Figure 2) than when the same samples were observed visually. qPCR was not used for samples with > 40% of the surface area necrotic because of the negative effect observed on the accuracy of analysis. The *M. graminicola* -tubulin gene on F3 was observed 24 weeks before symptoms appeared. There were five copies of the -tubulin gene per 100 ng leaf DNA (CBT100ng). The detection of *M. graminicola* DNA was two weeks later in the resistant cultivar than in the susceptible cultivar. The croissant gradient of the disease from the top of the plant to the bottom was also observed by qPCR analysis on all analysis dates except 2 and 8 June (GS 61 and 65 for Maxwell and GS 64 and 70 for Dinosor, respectively), when F1 qPCR values were higher than F2 and higher than or equal to F3. This increase was 14 days after the second period of the external contamination as determined by spore capture. This was characterized by the continuous arrival of ascospores (Figure 1b), combined with disease-favorable conditions, a high rate of precipitation (25 mm) and a temperature of 2025 C (Figure 1a).

#### **3.3 Wheat resistance to Septoria leaf blotch**

The resistance of wheat cultivars to *M. graminicola* was evaluated using visual symptoms assessment and qPCR analysis. Leaf samples were collected from the two leaf layers below the flag leaf, from the F3 leaf layer on 2 June at GS 61, 64, 65, 68 and 67, and from the F2 leaf layer on 16 June at GS 72, 71, 73, 73 and 73, of Maxwell, Dinosor, Alixan, Tremie and Maxyl, respectively. Results obtained for the *M. graminicola* DNA amount (Figure 3b), using either the F3 or F2 leaf layer, correlated well with the susceptibility rating given by the Arvalis Institut du Végétal (Arvalis Institut du Végétal 2008). Where two statistical groups were obtained, one presented the four susceptible cultivars (Dinosor, Alixan, Trémie and Maxyl) and the second presented the resistant cultivar (Maxwell). A close correlation was obtained between the results of the amount of DNA and the percentages of leaf necrotic area, especially when using the F2 leaf layer, where the discrimination between the two categories was clearer than with the F3 leaf layer (Figure 3a).

#### **3.4 Distribution of R-types of** *M. graminicola* **to DMI**

Quantification of the R-types of *M. graminicola* was not possible in the case of the Maxwell cultivar because of its low level of contamination. Therefore, the quantification of R-types (R6, R7, R7+ and I381) was realized only for the susceptible cultivars Dinosor, Alixan, Trémie and Maxyl. Analyses of R-types were carried out on two dates and using two leaf layers: F3 leaf samples were used on 2 June and F2 leaf samples on 16 June. For F3, the averages of CBT100ng were 1492, 2284, 1378 and 3606, and for F2 were they 3174, 5194, 3591 and 3642 for Dinosor, Alixan, Trémie and Maxyl, respectively. No significant differences were observed between R-types over the four cultivars used and over the two dates of analyses (Figure 3c). Regardless of the wheat cultivar, a high proportion of V381 strains ( 94%) was found, with an average of 74.2, 0.1 and 19.7% for R6, R7 and R7+ genotypes, respectively, whereas, I381-strains represent only 6% of all *M. graminicola* populations.

#### **3.5 Fungicide efficiency**

The effect of fungicide application on the development of Septoria blotch disease was investigated in an *in planta* experiment over 21 days. Within this period, the nongreen leaf area reached 15% on nontreated-inoculated control plants. Between 3 to 15 dpi, disease dynamics on control plants was slow (CBT100ng was 113–237 for Dinosor and 67–191 for Maxwell) but accelerated between 15 to 21 dpi to reach 1922 and 764 for Dinosor and Maxwell, respectively. The area under the disease progression curve (AUDPC) was calculated using qPCR analysis (CBT100ng) for the period from 3 to 21 dpi. For control plants, AUDPC was 8633 and 4614 for Dinosor and Maxwell, respectively. The AUDPC for all fungicide treatments was lower than in the control. Figure 4 shows a significant negative effect of all fungicide treatments on the development of fungal DNA regardless of the plant

after the second period of the external contamination as determined by spore capture. This was characterized by the continuous arrival of ascospores (Figure 1b), combined with disease-favorable conditions, a high rate of precipitation (25 mm) and a temperature of

The resistance of wheat cultivars to *M. graminicola* was evaluated using visual symptoms assessment and qPCR analysis. Leaf samples were collected from the two leaf layers below the flag leaf, from the F3 leaf layer on 2 June at GS 61, 64, 65, 68 and 67, and from the F2 leaf layer on 16 June at GS 72, 71, 73, 73 and 73, of Maxwell, Dinosor, Alixan, Tremie and Maxyl, respectively. Results obtained for the *M. graminicola* DNA amount (Figure 3b), using either the F3 or F2 leaf layer, correlated well with the susceptibility rating given by the Arvalis Institut du Végétal (Arvalis Institut du Végétal 2008). Where two statistical groups were obtained, one presented the four susceptible cultivars (Dinosor, Alixan, Trémie and Maxyl) and the second presented the resistant cultivar (Maxwell). A close correlation was obtained between the results of the amount of DNA and the percentages of leaf necrotic area, especially when using the F2 leaf layer, where the discrimination between the two categories

Quantification of the R-types of *M. graminicola* was not possible in the case of the Maxwell cultivar because of its low level of contamination. Therefore, the quantification of R-types (R6, R7, R7+ and I381) was realized only for the susceptible cultivars Dinosor, Alixan, Trémie and Maxyl. Analyses of R-types were carried out on two dates and using two leaf layers: F3 leaf samples were used on 2 June and F2 leaf samples on 16 June. For F3, the averages of CBT100ng were 1492, 2284, 1378 and 3606, and for F2 were they 3174, 5194, 3591 and 3642 for Dinosor, Alixan, Trémie and Maxyl, respectively. No significant differences were observed between R-types over the four cultivars used and over the two dates of analyses (Figure 3c). Regardless of the wheat cultivar, a high proportion of V381 strains ( 94%) was found, with an average of 74.2, 0.1 and 19.7% for R6, R7 and R7+ genotypes, respectively, whereas, I381-strains represent only 6% of all *M. graminicola*

The effect of fungicide application on the development of Septoria blotch disease was investigated in an *in planta* experiment over 21 days. Within this period, the nongreen leaf area reached 15% on nontreated-inoculated control plants. Between 3 to 15 dpi, disease dynamics on control plants was slow (CBT100ng was 113–237 for Dinosor and 67–191 for Maxwell) but accelerated between 15 to 21 dpi to reach 1922 and 764 for Dinosor and Maxwell, respectively. The area under the disease progression curve (AUDPC) was calculated using qPCR analysis (CBT100ng) for the period from 3 to 21 dpi. For control plants, AUDPC was 8633 and 4614 for Dinosor and Maxwell, respectively. The AUDPC for all fungicide treatments was lower than in the control. Figure 4 shows a significant negative effect of all fungicide treatments on the development of fungal DNA regardless of the plant

2025 C (Figure 1a).

populations.

**3.5 Fungicide efficiency** 

**3.3 Wheat resistance to Septoria leaf blotch** 

was clearer than with the F3 leaf layer (Figure 3a).

**3.4 Distribution of R-types of** *M. graminicola* **to DMI** 

cultivar. The most effective application occurred with the preventive treatment (d1), where fungal DNA was strongly decreased on all dates of observations: protection levels of 79% and 85% were achieved for Dinosor and Maxwell, respectively. Microscopic observations showed that spores failed to germinate when fungicide was applied one day before inoculation (Figure 5). Curative treatment (d+3) was similar to preventive treatment; the level of protection for Dinosor and Maxwell was 73 and 71%, respectively. Less fungicidal impact was observed when the fungicide was applied seven or ten days after inoculation (d+7 and d+10); the level of protection was 4560%.

Fig. 3. (a and b) Evaluation of cultivar resistance using visual symptoms observations and qPCR. (c) Frequency of DMI-resistant genotypes of *M. graminicola*; low resistant (I381) and moderate resistant (R6, R7 and R7+). (Leaf samples were collected from leaf layers F3 and F2 on 2 and 16 June 2009, respectively.)

Fig. 4. Prothioconazole efficiency against Septoria leaf blotch on Maxwell and Dinosor: d1, d+3, d+7 and d+10 are the fungicide treatments used one day before inoculation, and 3, 7 and 10 days after inoculation, respectively.

Fig. 5. Electron microscopic exposures of Dinosor leaves sampled at 15 dpi by a 10 l-drop of *M. graminicola* conidiospores 105.ml1: (a) nontreated control, where almost all spores germinated and stomata near from the point of inoculation were penetrated; and (b and c) wheat leaf treated with prothioconazole one day before inoculation (d1), where spores failed to germinate and almost all spores degraded.

#### **3.6 Validation of qPCR analyses**

228 Fungicides – Beneficial and Harmful Aspects

Fig. 4. Prothioconazole efficiency against Septoria leaf blotch on Maxwell and Dinosor: d1, d+3, d+7 and d+10 are the fungicide treatments used one day before inoculation, and 3, 7

Fig. 5. Electron microscopic exposures of Dinosor leaves sampled at 15 dpi by a 10 l-drop of *M. graminicola* conidiospores 105.ml1: (a) nontreated control, where almost all spores germinated and stomata near from the point of inoculation were penetrated; and (b and c) wheat leaf treated with prothioconazole one day before inoculation (d1), where spores

and 10 days after inoculation, respectively.

failed to germinate and almost all spores degraded.

#### **3.6.1 Relationship between qPCR analyses and** *S. tritici* **necrotic symptoms**

The second leaf layer (F2) of the susceptible cultivar Maxyl was used to study the correlation between DNA amounts measured by qPCR for leaf samples with different levels of necrotic surfaces, from 0 to 100% (Figure 6). *M. graminicola* DNA was detected in samples without symptoms and the DNA amount increased proportionally with an increase in leaf necrotic surface. A high level of correlation (CF = 0.95) was observed up to 40% leaf necrotic surface. The amount of *M. graminicola* DNA decreased beyond 50% leaf necrotic surface and remained stable.

Fig. 6. Linear relationship between necrotic symptoms and qPCR analysis for cultivar Maxyl (F2 leaf layer)

#### **3.6.2 Relationship between qPCR analysis and fungal colonization stages**

To understand the relationship between the amounts of *M. graminicola* DNA measured by qPCR and leaf colonization stage, a *M. graminicola*–wheat pathosystem with one point of inoculation was developed. The base part of the second leaf of 21-day-old plantlets (GS 13) was inoculated using 10 l (one drop) of *M. graminicola* inoculum (105 spores.ml1). Fungal colonization stages and the development of the amount of DNA are tabulated in Table 3.

One day after inoculation, 70% of spores had germinated and almost all germ tubes growing on the leaf surface were oriented toward the stomata (Figure 7a and b). Stomata located near the inoculation point were penetrated (Figure 7c and d). Appressorium-like structures (swellings of germ tube tips) were observed in contact with the ridges found at the guard cell lips (Figure 8a and b) and also sometimes over anticlinal walls, within the depressions between epidermal cells (Figure 8c, d and e). Electron microscopic observations of cryofractured leaf samples showed a direct penetration of leaf tissue in positions far from the stomata (Figure 8f). Intercellular colonization by mycelia growth was observed 5 and 7 dpi (Figure 7e). It was characterized by a progression from the inoculation point toward the top part of the leaf. This progression was fast at the beginning and became slower until 9 dpi. At 12 dpi, 55% of stomata were colored blue and cell death reached 20% of the leaf surface (Figure 7f). Disease development remained stable at this level until the appearance of symptoms at 26 dpi. This started from the leaf tip and progressed downward. Under the conditions used in this experiment, no pycnidium formation was observed by visual or microscopic observations.


\*Copy of *M. graminicola* -tubulin gene per gram of fresh leaf.

Table 3. Relationship between *M. graminicola* colonization stages and results of qPCR analysis

The qPCR assay was performed for samples collected on the same dates as for samples used for the microscopic observations. *M. graminicola* DNA evolution was characterized by an increase in the number of -tubulin gene copies per gram (CBTg) of fresh leaf weight from 327 at the day of inoculation to 683 copies at 12 dpi. The amount of DNA then remained stable over 10 days. At 26 dpi, during the appearance of symptoms, an increase in the amount of DNA becomes important.

#### **4. Discussion**

*Septoria* leaf blotch in wheat has a long latent period (Verreet et al., 2000). Prompt information about epidemic diseases, external contamination (arrival of ascospores) and fungicide-resistant alleles can be used to modify disease management strategies, based on the optimal use of host resistance, chemical control and cultural practices (Fraaije et al., 2005). In the present study, we highlight the importance of the use of qPCR in the study of Septoria leaf blotch epidemics.

Ascospores of *M. graminicola* present the primary disease inoculation. These could be produced all year round under specific conditions, such as certain weather conditions (Cordo et al., 1999), and by mature pseudothecia (Kema et al., 1996). Results of spore traps have indicated that external contamination by ascospores affects the *Septoria* leaf blotch epidemic by increasing the level of contamination in the upper leaf layers (F1 and F2).

between epidermal cells (Figure 8c, d and e). Electron microscopic observations of cryofractured leaf samples showed a direct penetration of leaf tissue in positions far from the stomata (Figure 8f). Intercellular colonization by mycelia growth was observed 5 and 7 dpi (Figure 7e). It was characterized by a progression from the inoculation point toward the top part of the leaf. This progression was fast at the beginning and became slower until 9 dpi. At 12 dpi, 55% of stomata were colored blue and cell death reached 20% of the leaf surface (Figure 7f). Disease development remained stable at this level until the appearance of symptoms at 26 dpi. This started from the leaf tip and progressed downward. Under the conditions used in this experiment, no pycnidium formation was observed by visual or

Day post inoculation Colonization state **CBTg\*** 0 Inoculation 327 1 Germination 367 3 Penetration 443

57 Internal colonization 533564

Table 3. Relationship between *M. graminicola* colonization stages and results of qPCR

The qPCR assay was performed for samples collected on the same dates as for samples used for the microscopic observations. *M. graminicola* DNA evolution was characterized by an increase in the number of -tubulin gene copies per gram (CBTg) of fresh leaf weight from 327 at the day of inoculation to 683 copies at 12 dpi. The amount of DNA then remained stable over 10 days. At 26 dpi, during the appearance of symptoms, an increase in the

*Septoria* leaf blotch in wheat has a long latent period (Verreet et al., 2000). Prompt information about epidemic diseases, external contamination (arrival of ascospores) and fungicide-resistant alleles can be used to modify disease management strategies, based on the optimal use of host resistance, chemical control and cultural practices (Fraaije et al., 2005). In the present study, we highlight the importance of the use of qPCR in the study of

Ascospores of *M. graminicola* present the primary disease inoculation. These could be produced all year round under specific conditions, such as certain weather conditions (Cordo et al., 1999), and by mature pseudothecia (Kema et al., 1996). Results of spore traps have indicated that external contamination by ascospores affects the *Septoria* leaf blotch epidemic by increasing the level of contamination in the upper leaf layers (F1 and F2).

12 Cells degradation 683 26 Symptoms (2%) 1785

\*Copy of *M. graminicola* -tubulin gene per gram of fresh leaf.

amount of DNA becomes important.

Septoria leaf blotch epidemics.

microscopic observations.

analysis

**4. Discussion** 

Fig. 7. Electron microscopic (a, c and e) and light microscopic (b, d and f) exposures of the colonization stages of Dinosor leaves inoculated by a 10 l-drop of *M. graminicola*  conidiospores (105.ml1): (a and b) spore germination at 1 dpi, 70% of spores germinated and almost of germ tubes grew on the leaf surface oriented toward the stomata; (c and d) at 3 dpi, stomata located near the inoculation point were penetrated; (e) at 5 and 7 dpi, the stomata were penetrated by passing through the stomatal guard cells and then intercellular colonization by mycelia growth; (f) at 12 dpi, 55% of the stomata were colored blue and cell death had reached 20% of the leaf surface. (Note the colonization of adjacent substomatal chambers and the surrounding tissue of the substomatal chamber.)

Fig. 8. Electron microscopic (a, b, c, e and f) and light microscopic (d) exposures of Dinosor leaves inoculated by a 10-l drop of *M. graminicola* conidiospores (105.ml1) sampled at 15 dpi: (a) stomata penetration by germ tubes; (b) two germ tubes with an appressorium-like structure penetrate a single stomata (arrows); (c, d and e) germ tube apical differentiations, which are similar to those seen over stomatal openings but here are associated with a leaf surface depression over an epidermal cell anticlinal wall (arrows); (f) cryofracture of *M. graminicola* colonized wheat leaf. Note that the site of penetration (arrowhead) is not per stomata, which proves the direct penetration by this fungus.

Fig. 8. Electron microscopic (a, b, c, e and f) and light microscopic (d) exposures of Dinosor leaves inoculated by a 10-l drop of *M. graminicola* conidiospores (105.ml1) sampled at 15 dpi: (a) stomata penetration by germ tubes; (b) two germ tubes with an appressorium-like structure penetrate a single stomata (arrows); (c, d and e) germ tube apical differentiations, which are similar to those seen over stomatal openings but here are associated with a leaf surface depression over an epidermal cell anticlinal wall (arrows); (f) cryofracture of *M. graminicola* colonized wheat leaf. Note that the site of penetration (arrowhead) is not per stomata, which proves the direct penetration by this

fungus.

The position of these leaf layers allows them to capture more air spores than the leaf layers at the bottom of the plant. This external contamination could affect fungicide efficiency by presenting a potential reinfection process, which perhaps coming out of the potential period of fungicide. Therefore, the detection of this reinfection source provides an actual interpretation of the efficiency of chemical treatment. In ascospore traps, the limitation of qPCR is the absence of specific gene markers that can discriminate between conidiospores and ascospores. However, *M. graminicola* conidia have a limited spread of only a few meters (Boeger et al., 1993), whereas ascospores spread over longer distances (Fraaije et al., 2005). Therefore, the limitation of qPCR was now successfully eliminated by removing all plants within a 3 m diameter of the traps in order to avoid the capture of rain-splash-dispersed conidia, as described in (Fraaije et al., 2005).

The accuracy of the use of qPCR to evaluate the resistance level of wheat cultivars to *M. graminicola* was investigated. Generally, results of qPCR analysis have a high correlation with the results of visual disease assessment (0.950.99). Detection of *M. graminicola* using qPCR was achieved 2–4 weeks before symptoms appeared and was 2 weeks earlier in the susceptible cultivars than in the resistant one. However, the *M. graminicola* epidemiology was well characterized for the resistant cultivar by a low level of contamination and a longer period of incubation than the susceptible cultivars. Generally, results of qPCR analyses of F3 or F2 DNA samples correlated well with the susceptibility rating of cultivars that were previously identified by the (Arvalis Institut du Végétal, 2008). A close correlation was obtained between qPCR results and the percentages of leaf necrotic area, especially with the F2 leaf layer. However, under natural infection conditions, visual assessment is suitable for assessing the combined resistance to all pathogens involved, but it is not suitable for assessing resistance to individual pathogens in a disease complex (Loughman et al., 1996). Visual assessment lacks accuracy and specificity and may be confused with other diseases, stress-related symptoms or even normal plant development ( Hollomon et al., 1999; Parker et al., 1995; Shimin, 2005). This problem was eliminated with qPCR, which has a high level of specificity of *M. graminicola* -tubulin and other microorganisms on the leaf surface that interfere with *M. graminicola* DNA are avoided (Fraaije et al., 1999). Furthermore, the using of multiplex qPCR (Fraaije et al., 2001), permits the quantification of other wheat pathogens in the same DNA sample.

The effect of wheat genotypes on the distribution of DMI-resistant R-types of *M. graminicola*  was also studied. As observed previously by (Selim, 2009), no significant effect of wheat cultivar was observed and the population structure was stable over all the analysis dates in the same season. The frequency of V381-genotypes was 90%, which agrees with the frequency of 50% reported in previous surveys carried out in 2005, 2006, 2007 and 2008 (Leroux et al., 2007; Stammler et al., 2008b), and 70% in 2009 (Selim, 2009). However, the presymptomatic detection and quantification of *M. graminicola* R-types is very important for the effective control of strains that are highly resistant to DMIs, as it allows better timing, choice and dose of fungicide applications. For example, genotypes R6, R7 and R7+ are less sensitive to tebuconazole but are sensitive to prochloraz, whereas genotypes carrying substitution V136A (R5) are most resistant to prochloraz (Fraaije et al., 2007; Leroux et al., 2007; Stammler et al., 2008a).

In the absence of total wheat cultivar resistance to *Septoria* leaf blotch, DMIs remain the key fungicide agents against *M. graminicola* (Leroux et al., 2007; Mavrolidi & Shaw, 2005). Their effects on *M. graminicola* are mostly attributed to the systemic action and inhibition of spore germination (Godwin et al., 1999). In the present study, qPCR analysis was used to evaluate the preventive and curative efficiency of DMI fungicides to control the more frequent genotypes of *M. graminicola* (R6, R7 and R7+). Results showed that prothioconazole treatments significantly decreased the amount of *M. graminicola* DNA by 80% in preventive treatment, and they were still 70% efficient when applied curatively at 3 dpi. Electron microscope observations showed that, after the preventive fungicide treatment (d1), the spores failed to germinate. Although all fungicide treatments resulted in a significant DNA reduction, compared with a nontreated control, later applications of prothioconazole, at 7 to 10 dpi, resulted in a high loss of efficiency ( 50%). These results are in agreement with those reported by (Godwin et al., 1999) and (Guo et al., 2007), namely that prothioconazole has a significant inhibitive effect against spore germination and postgermination, and could have a good curative effect against *M. graminicola* when applied at up to 20% of the latent period. However, most fungicides work best when applied early in the infection cycle, prior to visual symptom expression (O'Reilly et al., 1988). Thus, a qPCR monitoring process could help determine when fungicide application would be needed, and hence it could be determined when spraying would be most economical (Guo et al., 2006).

For a good understanding of the *M. graminicola* epidemic, cytological investigations are very important; however, under field conditions they are difficult to carry out (Kema et al., 1996). Therefore, we studied the relationship between qPCR analysis and the *M. graminicola* infection processes under controlled conditions and by using a one-drop inoculation method. This method is efficient because it controls the number of spores at the beginning of infection, it reduces points of penetration that delay disease development and it eliminates the limitation of the qPCR method, which arises from the DNA quantification of dead and nongerminated spores (Allmann et al., 1995; Josephson et al., 1993). However, penetration of leaves occurred mostly through the stomata, which is in agreement with previous reports (Cohen & Eyal 1993; Duncan & Howard, 2000; Kema et al., 1996; Shetty et al., 2003). Where, appressorium-like swellings were produced over the stomata as well as periclinally and anticlinally, and all stomatal penetration took place from germ tubes with swellings. Direct penetrations have been demonstrated previously (Cohen & Eyal, 1993; Dancer et al., 1999; Rohel et al., 2001; Shetty et al., 2003) and was also in rare cases. It has been suggested that it was due to a secondary mechanism of invasion of the host (Cohen & Eyal, 1993); however, its trigger factors are not known. The time course of the wheat infection processes of *M. graminicola* has been described by (Kema et al., 1996) and (Duncan & Howard, 2000). A strong correlation between the results of qPCR and microscopic observations was found, and disease development determined using qPCR had a similar pattern to that previously revealed by ELISA (Kema et al., 1996) or by PCR/PicoGreen assay (Fraaije et al., 1999). Until the formation of necrotic lesions, the biomass increased only slightly (or even decreased), but then increased rapidly during necrosis and the formation of pycnidia.

Our data confirm that qPCR is an accurate and specific method for studying *Septoria* leaf blotch epidemics in wheat, and for evaluating cultivar resistance, fungicide efficiency and the appearance of fungicide-resistant genotypes. Further research is required to improve our understanding of *M. graminicola* epidemics, taking in account wheat genotype resistance and the effects of fungicide application.

#### **5. Conclusion**

234 Fungicides – Beneficial and Harmful Aspects

Their effects on *M. graminicola* are mostly attributed to the systemic action and inhibition of spore germination (Godwin et al., 1999). In the present study, qPCR analysis was used to evaluate the preventive and curative efficiency of DMI fungicides to control the more frequent genotypes of *M. graminicola* (R6, R7 and R7+). Results showed that prothioconazole treatments significantly decreased the amount of *M. graminicola* DNA by 80% in preventive treatment, and they were still 70% efficient when applied curatively at 3 dpi. Electron microscope observations showed that, after the preventive fungicide treatment (d1), the spores failed to germinate. Although all fungicide treatments resulted in a significant DNA reduction, compared with a nontreated control, later applications of prothioconazole, at 7 to 10 dpi, resulted in a high loss of efficiency ( 50%). These results are in agreement with those reported by (Godwin et al., 1999) and (Guo et al., 2007), namely that prothioconazole has a significant inhibitive effect against spore germination and postgermination, and could have a good curative effect against *M. graminicola* when applied at up to 20% of the latent period. However, most fungicides work best when applied early in the infection cycle, prior to visual symptom expression (O'Reilly et al., 1988). Thus, a qPCR monitoring process could help determine when fungicide application would be needed, and hence it could be determined when spraying would be most

For a good understanding of the *M. graminicola* epidemic, cytological investigations are very important; however, under field conditions they are difficult to carry out (Kema et al., 1996). Therefore, we studied the relationship between qPCR analysis and the *M. graminicola* infection processes under controlled conditions and by using a one-drop inoculation method. This method is efficient because it controls the number of spores at the beginning of infection, it reduces points of penetration that delay disease development and it eliminates the limitation of the qPCR method, which arises from the DNA quantification of dead and nongerminated spores (Allmann et al., 1995; Josephson et al., 1993). However, penetration of leaves occurred mostly through the stomata, which is in agreement with previous reports (Cohen & Eyal 1993; Duncan & Howard, 2000; Kema et al., 1996; Shetty et al., 2003). Where, appressorium-like swellings were produced over the stomata as well as periclinally and anticlinally, and all stomatal penetration took place from germ tubes with swellings. Direct penetrations have been demonstrated previously (Cohen & Eyal, 1993; Dancer et al., 1999; Rohel et al., 2001; Shetty et al., 2003) and was also in rare cases. It has been suggested that it was due to a secondary mechanism of invasion of the host (Cohen & Eyal, 1993); however, its trigger factors are not known. The time course of the wheat infection processes of *M. graminicola* has been described by (Kema et al., 1996) and (Duncan & Howard, 2000). A strong correlation between the results of qPCR and microscopic observations was found, and disease development determined using qPCR had a similar pattern to that previously revealed by ELISA (Kema et al., 1996) or by PCR/PicoGreen assay (Fraaije et al., 1999). Until the formation of necrotic lesions, the biomass increased only slightly (or even decreased), but then increased rapidly during

Our data confirm that qPCR is an accurate and specific method for studying *Septoria* leaf blotch epidemics in wheat, and for evaluating cultivar resistance, fungicide efficiency and the appearance of fungicide-resistant genotypes. Further research is required to improve our understanding of *M. graminicola* epidemics, taking in account wheat genotype resistance and

economical (Guo et al., 2006).

necrosis and the formation of pycnidia.

the effects of fungicide application.

Close correlations were obtained between qPCR analysis and leaf colonization stages as well as with visual observations when the leaf had less than 40% necrotic area.

Real-time PCR results showed that late ascospore arrival increase the amount of *M. graminicola* DNA in the upper leaf layers (F1 and F2) and affect the real evaluation of fungicide efficiency. Distribution of DMI-resistant populations of *M. graminicola* was not affected by wheat cultivars. Cultivar resistance determined by qPCR correlated well with the susceptibility rating given by disease symptoms evaluation. Direct penetration of leaf tissue was confirmed by electron microscopy and, coupled with qPCR results, prothioconazole showed a significant inhibitive effect against spore germination and postgermination. We concluded that qPCR is an accurate and specific quantitative method for detecting and quantifying *M. graminicola* leaf blotch, in wheat, spore arrival, fungicide efficiency and fungicide-resistant genotypes, and for assessing the resistance of cultivars.

#### **6. Acknowledgments**

This work was financially supported by Bayer CropScience in France (BCSF).

#### **7. References**


Dancer, J., Daniels, A., Cooley, N., & Foster, S. 1999. *Septoria tritici* and *Stagonospora nodorum*

Duncan, K. E., & Howard, R. J. (2000). Cytological analysis of wheat infection by the leaf blotch pathogen *Mycosphaerella graminicola*. *Mycological Research*, *104*, 1074-1082. Fraaije, B. A., & Cools, H. J. (2008). Are azole fungicides losing ground against Septoria

Fraaije, B. A., Lowell, D. J., Rohel, E. A., & Hollomon, D. W. (1999). Rapid detection and

Fraaije, B. A., Cools, H. J., Kim, S. H., Motteram, J., Clark, W. S., & Lucas, J. A. (2007). A

Fraaije, B. A., Cools, H. J., Fountaine, J., Lovell, D. J., Motteram, J., West, J. S., & Lucas, J. A.

Godwin, J. R., Bartlett, D. W., & Heaney, S. P. 1999. Azoxystrobin: implications of biological

Guo, J. R., Schnieder, F., & Verreet, J. A. (2006). Presymptomatic and quantitative detection

Guo, J. R., Schnieder, F., & Verreet, J. A. (2007). A real-time PCR assay for quantitative and

Henson, J. M., & French, R. C. (1993). The polymerase chain reaction and plant disease

Hollomon, D. W., Fraaije, B., Rohel, E., Butters, J., & Kendall, S. 1999. Detection and

Josephson, K. L., Gerba, C. P., & Pepper, I. L. (1993). Polymerase chain reaction detection of

Kema, G. H. J., Yu, D., Rkenberg, F. H. J., Shaw, M. W., & Baayen, R. P. (1996). Histology of the pathogenesis of *Mycosphaerella graminicola* in wheat. *Phytopathology*, 86, 777-786

reaction/PicoGreen assay. *Journal of Applied Microbiology*, 86, 701-708 Fraaije, B. A., Lowell, D. J., Coelho, J. M., Baldwin, S., & Hollomon, D. W. (2001). PCR-based

UK: CABI Publishing

245-254

941

UK: CABI Publishing

UK: CABI Publishing.

*Management Science*, 64, 681-684

*Journal of Plant Pathology*, 107, 905-917

*FEMS Microbiology Letters*, 262, 223-229

*Journal of Phytopathology*, 155, 482-487

diagnosis. *Annual reviews of phytopathology*, 31, 81-109

as model pathogens for fungicide discovery. In *Septoria on cereals: a study of pathosystem*, edited by J. A. Lucas, P. Bowyer and H. M. Anderson. Wallingford,

wheat disease ? Resistance mechanisms in *Mycosphaerella graminicola*. *Pest* 

diagnosis of *Septoria tritici* epidemics in wheat using a polymerase chain

assay to assess wheat varietal resistance to blotch (*Septoria tritici* and *Stagonospora nodorum*) and rust (*Puccinia striiformis* and *Puccinia recondita*) diseases. *European* 

novel substitution I381V in the sterol 14 a-demethylase (CYP51) of *Mycosphaerella graminicola* is differentially selected by azole fungicides. *Molecular Plant Pathology*, 8,

(2005). Role of ascospores in further spread of QoI-resistant cytochrome *b* alleles (G143A) in field populations of *Mycosphaerella graminicola*. *Phytopathology*, 95, 933-

mode of action, pharmacokinetics and resistance management for spray programmes against Septoria diseases of wheat. In *Septoria on cereals: a study of pathosystems*, edited by J. A. Lucas, P. Bowyer and H. M. Anderson. Wallingford,

of *Mycosphaerella graminicola* development in wheat using a real-time PCR assay.

accurate assessment of fungicide effects on *Mycosphaerella graminicola* leaf blotch.

diagnosis of Septoria diseases: the problem in practice. In *Septoria on cereals: a study of pathosystems*, edited by J. A. Lucas, P. Bowyer and H. M. Anderson. Wallingford,

nonviable bacterial pathogens. *Applied and Environmental Microbiology*, 59, 3513-3515


### **Evaluation of Soybean (***Glycine max***) Canopy Penetration with Several Nozzle Types and Pressures**

Robert N. Klein, Jeffrey A. Golus and Greg R. Kruger *University of Nebraska WCREC, North Platte, NE, USA* 

#### **1. Introduction**

238 Fungicides – Beneficial and Harmful Aspects

Stammler, G., Carstensen, M., Koch, A., Semar, M., Strobel, D., & Schlehuber, S. (2008b).

Verreet, J. A., Klink, H., & Hoffmann, G. M. (2000). Regional monitoring for disease

*Plant Diseases*, 84, 816-826

Frequency of different CYP51-haplotypes of *Mycosphaerella graminicola* and their impact on epoxiconazole-sensivity and -field efficacy. *Crop Protection*, *27*, 1448-1456.

prediction and optimization of plant protection measures: the IPM wheat model.

Fungicides, when applied in the most effective way, can greatly improve the efficacy of the fungicide, reduce the risk of resistance, and potentially increase yields or preserve crops. When making fungicide applications, there are several things that must be considered. Most sprayers use hydraulic nozzles with pressure against an orifice. The applicator must consider which type of nozzle to use (both orifice size and nozzle type) as well as operating pressure.

Soybean rust is a foliar disease which has for many years been found mainly in Asian countries such as Taiwan, Japan, India, and more recently South Africa, Paraguay, Brazil, and Argentina (Dorrance, et al, 2009). *Phakopsora pachyrhizi* is one of the fungal species known to cause soybean rust and is the most aggressive. This species was identified in US soybean production fields in November of 2004. US cultivars are thought to be highly susceptible to this fungus, and efforts are underway to identify partial resistance or slow rusting traits. Fungicides have proven to be very effective in managing this disease and this will be the primary means of management for the first several years.

Soybean rust in the early days following infection can be found on the lower, first leaves of soybean plants (Geiseler, 2009). Therefore, to obtain control with fungicides one must penetrate the soybean canopy and get the fungicide down to where the infection occurs.

The objective of this study is to determine the optimum spray particle size that delivers the greatest coverage at lower levels of the soybean canopy.

Since the key to this research is the spray nozzle tip let's discuss spray nozzle tip technology.

The spray nozzle tip is important because it:


Where does one start in choosing a spray nozzle tip? The two important factors are pesticide efficacy and spray drift management.

Applicators want to use low water volumes to save time which makes coverage more of a concern. One needs knowledge of the product being used, whether it is a systemic or contact pesticide. Contact pesticides, such as paraquat, need more thorough coverage than systemic pesticides like glyphosate. Most fungicides and insecticides require thorough coverage which requires a smaller spray droplet size. Will this smaller droplet size penetrate the canopy? Also, what is the target? Is it soil, or grass, or broadleaf plants. Are the plant surfaces smooth, or hairy, or waxy? Leaf orientation and even the time of day of the application can affect the coverage needed and hence pesticide efficacy.

### **2. Nozzle description**

Nozzle types commonly used in low-pressure agricultural sprayers include: flat-fan, flood, raindrop, hollow-cone, full-cone, and others. Special features, or subtypes such as extended range, low pressure, drift guard, venturi-type and turbos are available for some nozzle types.

#### **2.1 Flat-fan**

Flat-fan nozzles, used for broadcast spraying, produce a tapered-edge spray pattern. These nozzles are also available for band spray. These nozzles are called even flat-fans, which produce a pattern with the same amount applied across the entire spray pattern. Other flatfan nozzle subtypes include the standard flat-fan, even flat-fan, low pressure flat-fan, extended range flat-fan, drift guards, Turbo TeeJet, and some special types such as off-center flat-fan and twin-orifice flat-fan.

The **standard** flat-fan normally is operated between 2.07 and 4.14 bar, with an ideal range 2.07 and 2.76 bar. The **even** (E) flat-fan nozzles (nozzle number ends with E) apply uniform coverage across the entire width of the spray pattern. They are used for **banding** pesticide over the row and should not be used for broadcast applications. The band width can be controlled with the nozzle height, spray angle, and the orientation of the nozzles.

The **low pressure** (LP) flat-fan develops a normal flat-fan angle and spray pattern at operating pressures between 1.03 and 1.72 bar. Lower pressures result in larger droplets and less drift, but a LP nozzle produces smaller droplets than a standard nozzle at the same pressure.

The **extended range** (XR or LFR) flat-fan provides excellent drift control when operated between 1.03 and 1.72 bar. This nozzle is ideal for an applicator who likes the uniform distribution of a flat-fan nozzle and desires lower operating pressure for drift control. Since extended range nozzles have an excellent spray distribution over a wide range of pressures 1.03 to 4.14 bar, they are ideal for sprayers equipped with flow controllers if spray particle drift is not a problem.

The **Turbo TeeJet** has the widest pressure range of the flat-fan nozzles - 1.03 to 6.21 bar. It produces larger droplets for less drift and is available only in 110 degree spray angle.

The **drift guard** flat-fan has a pre-orifice which controls the flow. The spray tip is approximately one nozzle size larger than the pre-orifice and therefore produces larger droplets and reduces the small drift prone droplets.

Where does one start in choosing a spray nozzle tip? The two important factors are pesticide

Applicators want to use low water volumes to save time which makes coverage more of a concern. One needs knowledge of the product being used, whether it is a systemic or contact pesticide. Contact pesticides, such as paraquat, need more thorough coverage than systemic pesticides like glyphosate. Most fungicides and insecticides require thorough coverage which requires a smaller spray droplet size. Will this smaller droplet size penetrate the canopy? Also, what is the target? Is it soil, or grass, or broadleaf plants. Are the plant surfaces smooth, or hairy, or waxy? Leaf orientation and even the time of day of the

Nozzle types commonly used in low-pressure agricultural sprayers include: flat-fan, flood, raindrop, hollow-cone, full-cone, and others. Special features, or subtypes such as extended range, low pressure, drift guard, venturi-type and turbos are available for some nozzle

Flat-fan nozzles, used for broadcast spraying, produce a tapered-edge spray pattern. These nozzles are also available for band spray. These nozzles are called even flat-fans, which produce a pattern with the same amount applied across the entire spray pattern. Other flatfan nozzle subtypes include the standard flat-fan, even flat-fan, low pressure flat-fan, extended range flat-fan, drift guards, Turbo TeeJet, and some special types such as off-center

The **standard** flat-fan normally is operated between 2.07 and 4.14 bar, with an ideal range 2.07 and 2.76 bar. The **even** (E) flat-fan nozzles (nozzle number ends with E) apply uniform coverage across the entire width of the spray pattern. They are used for **banding** pesticide over the row and should not be used for broadcast applications. The band width can be

The **low pressure** (LP) flat-fan develops a normal flat-fan angle and spray pattern at operating pressures between 1.03 and 1.72 bar. Lower pressures result in larger droplets and less drift, but a LP nozzle produces smaller droplets than a standard nozzle at the same pressure.

The **extended range** (XR or LFR) flat-fan provides excellent drift control when operated between 1.03 and 1.72 bar. This nozzle is ideal for an applicator who likes the uniform distribution of a flat-fan nozzle and desires lower operating pressure for drift control. Since extended range nozzles have an excellent spray distribution over a wide range of pressures 1.03 to 4.14 bar, they are ideal for sprayers equipped with flow controllers if spray particle

The **Turbo TeeJet** has the widest pressure range of the flat-fan nozzles - 1.03 to 6.21 bar. It produces larger droplets for less drift and is available only in 110 degree spray angle.

The **drift guard** flat-fan has a pre-orifice which controls the flow. The spray tip is approximately one nozzle size larger than the pre-orifice and therefore produces larger

controlled with the nozzle height, spray angle, and the orientation of the nozzles.

application can affect the coverage needed and hence pesticide efficacy.

efficacy and spray drift management.

**2. Nozzle description** 

flat-fan and twin-orifice flat-fan.

drift is not a problem.

droplets and reduces the small drift prone droplets.

types.

**2.1 Flat-fan** 

The **venturi-type** nozzle produces large air-filled drops through the use of a venturi air aspirator for reducing drift. These include the Delavan Raindrop Ultra, Greenleaf TurboDrop, Lurmark Ultra Lo-Drift, Spraying Systems AI Teejet, ABJ Agri. Products Air Bubble Jet, and Wilger's Combo-Jet. Some of these nozzle tips are available in extended range for pressures.

Flat-fan nozzles also include the **off-center** (LX) flat-fan which is used for boom end nozzles so a wide swath projection is obtained and the **twin-orifice** (TJ) flat-fan which produces two spray patterns -- one angled 30 degrees forward and the other directed 30 degrees backwards. The TJ droplets are small because the spray volume is passing through two smaller orifices instead of one larger one. The two spray directions and smaller droplets improve coverage and penetration, a plus when applying postemergence contact herbicides. To produce fine droplets, the twin-orifice usually operates between 2.07 and 4.14 bar.

Flat-fan nozzles are available in several spray angles. The most common spray angles are 65, 73, 80, and 110 degrees. Recommended nozzle heights for flat-fan nozzles during broadcast application are given in Table 1.



Table 1. Suggested minimum spray heights.

The correct nozzle height is measured from the nozzles to the target, which may be the top of the ground, growing canopy, or stubble. Use 110 degree nozzles when boom heights are less than 76 cm and 80 degree nozzles when the booms are higher.

Although wide-angle nozzles produce smaller droplets that may be more prone to drift, the reduction of boom height reduces the overall drift potential. The net reduction in drift potential more than offsets the effect of smaller droplet size. The nozzle spacing and orientation should provide for 100 percent overlap at the target height. Nozzles should not be oriented more than 30 degrees from vertical.

Most nozzle manufacturers identify their flat-fan nozzles with a four or five digit number. The first numbers are the spray angle and the other numbers signify the discharge rate at rated pressure. For example, an 8005 has an 80 degree spray angle and will discharge 1.9 liters per minute (LPM) at the rated pressure of 2.75 bar. A 11002 nozzle has a 110 degree spray angle and will discharge 0.8 LPM at the rated pressure of 2.75 bar. Additional designations are: " BR" - brass material; "SS" - stainless steel; "VH" - hardened stainless steel; and "VS" - color codes. See Table 2 for nozzle type and discharge rates.


Table 2. Nozzles types and discharge rates at rated pressure.

Delavan flat-fan nozzles are identified by LF or LF-R, which reflect the standard and extended range flat-fan nozzles. The first numbers are the spray angle followed by a dash, and then the discharge rate at rated pressure. For example, an LF80-5R is an extended range nozzle with an 80 degree spray angle, and will apply 1.9 LPM at the rated pressure of 2.75 bar.

#### **2.2 Flood**

Flood nozzles are popular for applying suspension fertilizers where clogging is a potential problem. These nozzles produce large droplets at pressures of 0.69 to 1.72 bar. The nozzles should be spaced less than 152 cm apart. The nozzle height and orientation should be set for 100 percent overlap.

Nozzle spacing between 76 and 102 cm produces the best spray patterns. Pressure influences spray patterns of flood nozzles more than flat-fan nozzles. However, the spray pattern is not as uniform as with the flat-fan nozzles, and special attention to nozzle orientation and correct overlap is critical.

Flooding nozzles are designated "TK" by Spraying Systems and "D" by Delavan. The value following the letters is the flow rate at the rated pressure of 0.69 bar. For example, TK-SS2 or D-2 are flood nozzles that apply 0.8 LPM at 0.7 bar.

The Turbo FloodJet incorporates a pre-orifice which controls the flow plus a turbulence chamber. The tip design more closely resembles a flat fan nozzle, which greatly reduces the surface area and the result is a much improved pattern with tapered edges. Use the turbo flood for pesticide application for incorporated herbicides because of the improved pattern. The turbo flood nozzles are a good choice if drift is a concern because they produce larger droplets than standard flood nozzles. Because of their large droplet size do not use the turbo flood nozzle where good coverage is needed.

#### **2.3 Raindrop**

242 Fungicides – Beneficial and Harmful Aspects

Rated Pressure (bar)

Operating Range

Min (bar) Max (bar)

(lpm)

Regular flat-fan 8006 2.3 2.76 2.07 4.14 Regular flat-fan 11008 3.0 2.76 2.07 4.14 Low pressure flat-fan 8006LP 2.3 1.03 1.03 2.76 Low pressure flat-fan 11008LP 3.0 1.03 1.03 2.76 Extended range flat-fan 8006XR 2.3 2.76 1.03 4.14 Extended range flat-fan 11008XR 3.0 2.76 1.03 4.14 Turbo TeeJet TT11002VP 0.8 2.76 1.03 6.21 Turbo TeeJet TT11005VP 1.9 2.76 1.03 6.21 Turbo TeeJet Induction TTI11002 0.8 1.03 2.76 6.89 Turbo TeeJet Induction TTI11005 1.9 1.03 2.76 6.89 Drift Guard DG8002VS 0.8 2.76 2.76 4.14 Drift Guard DG11005VS 1.9 2.76 2.07 4.14 AI TeeJet AI11002-VS 0.8 2.76 2.07 6.89 AI TeeJet AI11005-VS 1.9 2.76 2.07 6.89 AIXR TeeJet 11002VS 0.8 2.76 1.03 6.21 AIXR TeeJet 11005VS 1.9 2.76 1.03 6.21 Flood TKSS 6 2.3 0.69 0.69 2.76 Flood TKSS 8 3.0 0.69 0.69 2.76 Turbo FloodJet TF-VS2 0.8 0.69 0.69 2.76 Turbo FloodJet TF-VS10 3.8 0.69 0.69 2.76 Raindrop RA-6 2.3 2.76 1.38 3.45

Nozzle Type Discharge

Table 2. Nozzles types and discharge rates at rated pressure.

**2.2 Flood** 

100 percent overlap.

orientation and correct overlap is critical.

D-2 are flood nozzles that apply 0.8 LPM at 0.7 bar.

Delavan flat-fan nozzles are identified by LF or LF-R, which reflect the standard and extended range flat-fan nozzles. The first numbers are the spray angle followed by a dash, and then the discharge rate at rated pressure. For example, an LF80-5R is an extended range nozzle with an

Flood nozzles are popular for applying suspension fertilizers where clogging is a potential problem. These nozzles produce large droplets at pressures of 0.69 to 1.72 bar. The nozzles should be spaced less than 152 cm apart. The nozzle height and orientation should be set for

Nozzle spacing between 76 and 102 cm produces the best spray patterns. Pressure influences spray patterns of flood nozzles more than flat-fan nozzles. However, the spray pattern is not as uniform as with the flat-fan nozzles, and special attention to nozzle

Flooding nozzles are designated "TK" by Spraying Systems and "D" by Delavan. The value following the letters is the flow rate at the rated pressure of 0.69 bar. For example, TK-SS2 or

80 degree spray angle, and will apply 1.9 LPM at the rated pressure of 2.75 bar.

Raindrop nozzles produce large drops in a hollow-cone pattern at pressures from 1.38 to 3.45 bar. The "RA" Raindrop nozzles are used for pre-plant incorporated herbicide and are usually mounted on tillage implements. When used for broadcast application, nozzles should be orientated 30 degrees from the horizontal. The spray patterns should be overlapped 100 percent to obtain uniform distribution. These nozzles are not satisfactory for postemergence or non-incorporated herbicides because the small number of large droplets produced would not provide satisfactory coverage.

#### **2.4 Cone**

**Hollow-cone** - Hollow cone nozzles generally are used to apply insecticides or fungicides to field crops when foliage penetration and complete coverage of the leaf surface is required. These nozzles operate in a pressure range from 2.76 to 6.89 bar. Spray drift potential is higher from hollow-cone nozzles than from other nozzles due to the small droplets produced.

**Full-cone** - Full-cone nozzles usually are recommended over flood nozzles for soilincorporated herbicides. Full-cone nozzles operate between a pressure range of 1.03 to 2.76 bar. Optimum uniformity is achieved by angling the nozzles 30 degrees and overlapping the spray coverage by 100 percent.

**Fine Hollow-cone** - The ConeJet (Spray Systems) and WRW-Whirl Rain (Delavan) are wideangle (80 to 120 degrees), hollow-cone nozzles. These nozzles are used for postemergence contact herbicides where a finely atomized spray is used for complete coverage of plants or weeds under a hood for band spraying. Drift potential is great for these nozzles.

#### **2.5 Nozzle material**

Nozzles can be made from several materials. The most common are brass, nylon, stainless steel and hardened stainless steel and ceramic. Stainless steel nozzles last longer than brass or nylon and generally produce a more uniform pattern over an extended time period. Nylon nozzles with stainless steel or hardened stainless steel inserts offer an alternative to solid stainless steel nozzles at a reduced cost. Thermoplastic nozzles have good abrasion resistance but swelling can occur with some chemicals, and they are easily damaged when cleaned. Ceramic has superior wear life and is highly resistant to abrasive and erosive chemicals. Where available ceramic is usually the best choice.

Do not mix nozzles of different materials, types, spray angles, or spray volumes on the same spray boom. A mixture of nozzles produces uneven spray distribution.

#### **2.6 Combination nozzles**

These are where the spray tip is built right into the cap as one. These keep the spray tip and cap from separating and when available is usually the best choice.

#### **2.7 When to replace nozzles**


**Note:** Each nozzle's flow rate on spray boom needs to be within 5% of the average nozzle flow rate.

Spray particle size affects both pesticide efficacy (coverage) and spray drift. Cutting the droplet size in half results in eight times the number of spray droplets, see Figure 1.

Fig. 1. Reducing droplet size by 50% results in eight times the number of droplets

The origin of standardization of spray droplet sizes started with the British Crop Protection Council in 1985 with droplet size classification, primarily designed to enhance efficacy. It uses the term, *SPRAY QUALITY* for droplet size categories. In 2000 the ASAE Standard S572 established the droplet size classification in the U.S. primarily designed to control spray drift and secondarily efficacy. It uses the term *DROPLET SPECTRA CLASSIFICATION* for droplet size categories. In March 2009 ANSI/ASAE approved S572.1 as the American National Standard. This added extremely fine and ultra coarse to the classification categories.

The specifics of the Standard ASAE S572.1.


Important Droplet Characteristics:

244 Fungicides – Beneficial and Harmful Aspects

These are where the spray tip is built right into the cap as one. These keep the spray tip and

**Note:** Each nozzle's flow rate on spray boom needs to be within 5% of the average nozzle

Spray particle size affects both pesticide efficacy (coverage) and spray drift. Cutting the

**500 microns**

> **250 microns**

**250 microns**

**250 microns**

**250 microns**

droplet size in half results in eight times the number of spray droplets, see Figure 1.

**250 microns**

Fig. 1. Reducing droplet size by 50% results in eight times the number of droplets

The origin of standardization of spray droplet sizes started with the British Crop Protection Council in 1985 with droplet size classification, primarily designed to enhance efficacy. It uses the term, *SPRAY QUALITY* for droplet size categories. In 2000 the ASAE Standard S572 established the droplet size classification in the U.S. primarily designed to control spray drift and secondarily efficacy. It uses the term *DROPLET SPECTRA CLASSIFICATION* for droplet size categories. In March 2009 ANSI/ASAE approved S572.1 as the American National Standard. This added extremely fine and ultra coarse to the classification


**250 microns**

cap from separating and when available is usually the best choice.


**2.6 Combination nozzles** 

**2.7 When to replace nozzles**  - Spray pattern distorted - Nozzles show irregular wear

> **250 microns**

**250 microns**

The specifics of the Standard ASAE S572.1.




flow rate.

categories.

condition.

Dv0.1(µm) - 10% of spray volume is of droplet sizes less than this number Dv0.5(µm) - 50% of spray volume is of droplet sizes less than this number; volume median diameter (VMD)

Dv0.9(µm) - 90% of spray volume is of droplet sizes less than this number

Though not part of the standard, the percent of spray volume less than 200 microns identifies the particle sizes most prone to spray drift.

Figures 2 and 3, and Table 3 illustrate volume median diameter, the ASAE Standard and a Turbo TeeJet spray tip droplet spectra.

Fig. 2. Volume Median Diameter (VMD)

Fig. 3. ANSI/ASAE S572.1 March 2009; Approved as an American National Standard



Three nozzle tips were evaluated for the control of triazine resistant kochia and green foxtail in winter wheat stubble. The treatment parameters used to evaluate the nozzle tips and percent control are shown in Table 4 and Figures 4 and 5.


\*All treatments applied at 2 bar

\*Herbicide applied was Paraquat + Atrazine (0.35 + 0.56 Kg ai/ha)

Table 4. Treatment parameters used to evaluate three nozzle types

TT11001 C M M M M M F F F F F

TT110015 C C M M M M M M F F F

TT11002 C C C M M M M M M M F

TT11003 VC VC C C C C M M M M M

TT11004 XC VC VC C C C C C M M M

TT11005 XC VC VC VC VC C C C C M M

TT11006 XC XC VC VC VC C C C C C M

TT11008 XC XC VC VC VC VC C C C C M

Three nozzle tips were evaluated for the control of triazine resistant kochia and green foxtail in winter wheat stubble. The treatment parameters used to evaluate the nozzle tips and

1 XR11005 Coarse 10 94 8.6 14 2 DG11005 Coarse 10 94 8.6 14 3 TF-VS2.5 Extremely Coarse 10 94 8.6 14 4 XR11004 Medium 7.5 70 9.2 15 5 DG11004 Coarse 7.5 70 9.2 15 6 TF-VS2 Extremely Coarse 7.5 70 9.2 15 7 XR11003 Fine 5.0 47 10.3 17 8 DG11003 Coarse 5.0 47 10.3 17

Check --- ---

**Volume Speed** 

(gpa) (L/ha) (mph) (km/h)

Table 3. Turbo TeeJet® Nozzle Droplet Spectra

**Trt Nozzle Spray Particle** 

<sup>9</sup>Untreated

\*All treatments applied at 2 bar

percent control are shown in Table 4 and Figures 4 and 5.

**Size** 

\*Herbicide applied was Paraquat + Atrazine (0.35 + 0.56 Kg ai/ha)

Table 4. Treatment parameters used to evaluate three nozzle types

1.03 1.38 1.72 2.07 2.41 2.76 3.45 4.14 4.83 5.52 6.21

Nozzle Bar

Fig. 4. Triazine resistant kochia control with several nozzles and carrier volumes

Fig. 5. Green foxtail control with several nozzles and carrier volumes

The results of a study on the retention and total uptake and root translocation of glyphosate of glyphosate resistant corn by Paul Feng of Monsanto are in Table 5 and Figure 6.


Table 5. Effect of droplet size on glyphosate retention in glyphosate resistant corn

Fig. 6. Uptake and translocation in glyphosate resistant corn with fine, medium and coarse spray droplets.

In the glyphosate resistant corn study even though there was less glyphosate retained on the plants with the coarse droplet size as compared to the fine, the coarse droplet size resulted in increased uptake and root translocation. It is thought that there must be enough glyphosate present in the spray droplet to translocate in the plant.

A study across Nebraska in 2004 with five nozzle tips resulted in almost identical weed control. One should therefore select those tips which produce the smallest amount of fines which are the Turbo TeeJet, Turbo Flood and Air Induction nozzle tips. See Figure 7.

Figure 8 gives the particle sizes of five nozzle tips with water and water with Roundup WeatherMax + 2% Ammonium Sulfate and three additives: Array, In-Place and Interlock.

The results of a study on the retention and total uptake and root translocation of glyphosate

% Retention

0

0 0.5 1 1.5 2 2.5 3 3.5 **DAT after Roundup spray (32 oz/a)**

5

10

**(Avg 4 + SE)**

**Root as % recov'd dose** 

**48.9% 35.1% 30.1%**

Fig. 6. Uptake and translocation in glyphosate resistant corn with fine, medium and coarse

In the glyphosate resistant corn study even though there was less glyphosate retained on the plants with the coarse droplet size as compared to the fine, the coarse droplet size resulted in increased uptake and root translocation. It is thought that there must be enough

A study across Nebraska in 2004 with five nozzle tips resulted in almost identical weed control. One should therefore select those tips which produce the smallest amount of fines

Figure 8 gives the particle sizes of five nozzle tips with water and water with Roundup WeatherMax + 2% Ammonium Sulfate and three additives: Array, In-Place and Interlock.

which are the Turbo TeeJet, Turbo Flood and Air Induction nozzle tips. See Figure 7.

15

**Coarse Medium Fine**

20

(Actual over Calculated)

of glyphosate resistant corn by Paul Feng of Monsanto are in Table 5 and Figure 6.

Table 5. Effect of droplet size on glyphosate retention in glyphosate resistant corn

Fine 47 ± 2 Medium 37 ± 7 Coarse 38 ± 8

0 0.5 1 1.5 2 2.5 3 3.5 **DAT after Roundup spray (32 oz/a)**

glyphosate present in the spray droplet to translocate in the plant.

**Coarse Medium Fine**

Droplet Size

spray droplets.

**Uptake as % recov'd dose** 

**(Avg 4 + SE)**

Fig. 7. Percent visual control of corn, oil sunflower, velvetleaf, green foxtail and watermemp with glyphosate.

Fig. 8. Volume median diameter of several nozzles and spray solutions.

The volume median diameter of the spray particle sizes are greatly reduced as was the amount of spray volume under 210 microns with the addition of Roundup WeatherMax and 2% AMS. The additive Array increased the volume median diameter and reduced the amount of spray volume under 210 microns with the extended range, Turbo TeeJet and Turbo Flood nozzles. The additives In-Place and Interlock performed with similar results with the air induction and extended range air induction tip. All additives do not work with all nozzles as evident in Figure 9.

To evaluate the nozzle tips, pressure, nozzle spacing and angle in getting penetration into the soybean canopy, research was conducted over several years. Soybeans were planted in 76.2 cm rows in May 2006, 2007, and 2008. Field applications were conducted in August and September of these years. Six different nozzles were included, all Spraying Systems Co: XRC11003, XRC11006, TT11003, TT11006, AIC110025, and AIC11005. Each nozzle was used at three pressures, and two different nozzle setups were included. Nozzles were set on 76.2 cm spacing, and 190 l ha-1 was the carrier volume (Table 6). For the smaller nozzle size of each type, two nozzles were used: one directed 45 degrees forward from vertical and the other directed 45 degrees back from vertical. Boom height was 43.2 cm above the canopy. White indicating cards were set into a row of soybeans. The cards were attached to an electric fencepost at heights of 14 cm (low), 42 cm (middle) and 70 cm (high), with the soybeans being 84 cm tall. A pull type sprayer was used to apply the treatments. Water dyed with Garrco Products Vision Pink indicating dye was sprayed over the cards. Four sets of cards were placed for each treatment to create four replications. A nozzle setup ran directly over the row of soybeans containing the cards. The cards were allowed to dry and placed in Ziploc bags. The cards were then scanned with the program DropletScan, which determines the number of drops, volume median diameter (VMD) and percent coverage for each card. VMD is the micron size of which half the spray volume in made of smaller droplets and half is of larger droplets.


Table 6. Nozzles and pressures used in soybean canopy penetration study.

The nozzles were also analyzed with a Sympatec Helos Vario KF particle size analyzer. With the R6 lens installed, it is capable of detecting particle sizes in a range from 0.5 to 1550 microns. This system uses laser diffraction to determine particle size distribution. Each treatment was replicated three times. The width of the nozzle plume was analyzed by moving the nozzle across the laser by means of a linear actuator. Information obtained includes VMD.

#### **3. Results**

250 Fungicides – Beneficial and Harmful Aspects

The volume median diameter of the spray particle sizes are greatly reduced as was the amount of spray volume under 210 microns with the addition of Roundup WeatherMax and 2% AMS. The additive Array increased the volume median diameter and reduced the amount of spray volume under 210 microns with the extended range, Turbo TeeJet and Turbo Flood nozzles. The additives In-Place and Interlock performed with similar results with the air induction and extended range air induction tip. All additives do not work with

To evaluate the nozzle tips, pressure, nozzle spacing and angle in getting penetration into the soybean canopy, research was conducted over several years. Soybeans were planted in 76.2 cm rows in May 2006, 2007, and 2008. Field applications were conducted in August and September of these years. Six different nozzles were included, all Spraying Systems Co: XRC11003, XRC11006, TT11003, TT11006, AIC110025, and AIC11005. Each nozzle was used at three pressures, and two different nozzle setups were included. Nozzles were set on 76.2 cm spacing, and 190 l ha-1 was the carrier volume (Table 6). For the smaller nozzle size of each type, two nozzles were used: one directed 45 degrees forward from vertical and the other directed 45 degrees back from vertical. Boom height was 43.2 cm above the canopy. White indicating cards were set into a row of soybeans. The cards were attached to an electric fencepost at heights of 14 cm (low), 42 cm (middle) and 70 cm (high), with the soybeans being 84 cm tall. A pull type sprayer was used to apply the treatments. Water dyed with Garrco Products Vision Pink indicating dye was sprayed over the cards. Four sets of cards were placed for each treatment to create four replications. A nozzle setup ran directly over the row of soybeans containing the cards. The cards were allowed to dry and placed in Ziploc bags. The cards were then scanned with the program DropletScan, which determines the number of drops, volume median diameter (VMD) and percent coverage for each card. VMD is the micron size of which half the

spray volume in made of smaller droplets and half is of larger droplets.

bar

1.03 2.07 4.14

1.03 2.07 4.14

1.03 2.07 4.14

1.03 2.07 4.14

2.07 4.14 6.21

2.07 4.14 6.21

Table 6. Nozzles and pressures used in soybean canopy penetration study.

Total Nozzle Output L min-1

> 1.36 1.97 2.80

> 1.36 1.97 2.80

> 1.36 1.97 2.80

> 1.36 1.97 2.80

> 1.67 2.35 3.88

> 1.67 2.35 3.88

Speed km hr-1

> 05.8 08.2 11.8

> 05.8 08.2 11.8

> 05.8 08.2 11.8

> 05.8 08.2 11.8

> 05.8 08.2 11.8

> 05.8 08.2 11.8

Treatment Nozzle(s) Pressure

XRC 11003 (2)

XRC 11006

TT 11003 (2)

TT 11006

AIC 110025 (2)

AIC 11005

1 2 3

4 5 6

7 8 9

10 11 12

13 14 15

16 17 18

all nozzles as evident in Figure 9.

The results of coverage on the cards scanned with the program DropletScan are reported in Table 7. This includes the three pressures, VMD and the percent coverage on the lower, middle and upper cards. Also listed in the table are the laser VMD for each nozzle and pressure. Results are from 2006, 2007 and 2008 combined.


aVolume median diameter

bPercent coverage

Table 7. Volume median diameter (VMD) and percent coverage for various nozzles, pressures and card placement in soybean canopy.

#### **4. Discussion**

The figures 9, 10 and 11 contain results of VMD and percent coverage of the card in the three card levels in soybean canopy for the 3 years, 2006 to 2008. Data in the figures represents the average of the three pressures used, while Table 7 includes data for each pressure. With the lower card, the TT11003 had the best coverage closely followed by the TT11006. With the XRC nozzles, as the pressure increased the coverage decreases in the lower canopy. This was also true with the smaller TT (2-TT11003 in opposite directions) but the larger TT spraying straight down and the AIC nozzle coverage decreased with the medium pressure but increased again with the highest pressure. The two TT11003 had the highest amount of coverage of the three nozzle types in the study.

For the lower card, laser VMD was larger than card VMD for each nozzle. As particle size increased (comparing nozzle types), the difference between the VMD's increased, especially at laser VMD sizes of 375 µm and greater. For the middle card, the two VMD's were equal up to 375 µm. Above this point, laser VMD once again becomes larger. For the upper card, card VMD was greater than laser VMD up to laser VMD being 450 µm. Above that, card VMD was smaller. This suggests larger particles landed in the upper canopy, especially for nozzles producing a smaller VMD (smaller particle size). The nozzles producing the highest percent coverage of the lower card were the TT11003 and TT11006. These nozzles produced a laser VMD of 383 µm and 464 µm respectively, suggesting a micron size of around 400 may be optimal.

Fig. 9. VMD and percent coverage of lower card in soybean canopy

The figures 9, 10 and 11 contain results of VMD and percent coverage of the card in the three card levels in soybean canopy for the 3 years, 2006 to 2008. Data in the figures represents the average of the three pressures used, while Table 7 includes data for each pressure. With the lower card, the TT11003 had the best coverage closely followed by the TT11006. With the XRC nozzles, as the pressure increased the coverage decreases in the lower canopy. This was also true with the smaller TT (2-TT11003 in opposite directions) but the larger TT spraying straight down and the AIC nozzle coverage decreased with the medium pressure but increased again with the highest pressure. The two TT11003 had the highest amount of

For the lower card, laser VMD was larger than card VMD for each nozzle. As particle size increased (comparing nozzle types), the difference between the VMD's increased, especially at laser VMD sizes of 375 µm and greater. For the middle card, the two VMD's were equal up to 375 µm. Above this point, laser VMD once again becomes larger. For the upper card, card VMD was greater than laser VMD up to laser VMD being 450 µm. Above that, card VMD was smaller. This suggests larger particles landed in the upper canopy, especially for nozzles producing a smaller VMD (smaller particle size). The nozzles producing the highest percent coverage of the lower card were the TT11003 and TT11006. These nozzles produced a laser VMD of 383 µm and 464 µm respectively, suggesting a micron size of around 400

**4. Discussion** 

may be optimal.

coverage of the three nozzle types in the study.

Fig. 9. VMD and percent coverage of lower card in soybean canopy

Fig. 10. VMD and percent coverage of middle card in soybean canopy

Fig. 11. VMD and percent coverage of upper card in soybean canopy

#### **5. References**


Dorrance, Anne E., Patrick E. Lipps, Dennis Mills and Miguel Vega-Sanchez. 2009. Soybean

Geiseler, Loren J. 2009. Asian Soybean Rust. University of Nebraska-Lincoln Plant Disease

Klein, Robert N., Jeffrey A. Golus, Alexander R. Martin, Fred W. Roeth, and Brady F.

Klein, Robert, Jeffrey Golus and Amanda Cox. 2008. Spray Droplet Size and How It Is

Klein, Robert and Jeffrey Golus. 2010. Evaluation of Soybean (*Glycine max*) Canopy

Klein, Robert N. 2010. Spray Nozzle Tip Technology. pp. 195-200. Proceedings 2010 Crop

Feng, Paul C.C., Tommy Chiu, R. Douglas Sammons, and Jan S. Ryerse. 2003. Droplet Size

Production Clinics, University of Nebraska-Lincoln-Extension.

Central. University of Nebraska-Lincoln, Lincoln NE.

Robinson College, Cambridge, UK, January 9-11, 2008.

Rust. Ohio State University Extension Fact Sheet AC-0048-04. Ohio State

Kappler. 2006. Glyphosate Efficacy With Air Induction, Extended Range, Turbo FloodJet and Turbo TeeJet Nozzle Tips. pp. 453-460. International Advances in Pesticide Application 2006, Robinson College, Cambridge, UK, January 10-12, 2006.

Affected by Pesticide Formulation, Concentrations, Carrier, Nozzle Tips, Pressure and Additives. pp. 232-238. International Advances in Pesticide Application 2006,

Penetration With Several Nozzle Types and Pressure. pp. 35-39. 2010. International Advances in Pesticide Application 2006, Robinson College, Cambridge, UK,

Affects Glyphosate Retention, Absorption, and Translocation in Corn. Weed

**5. References** 

University, Columbus Ohio.

January 5-7, 2010

Science, 51:443-448.

### *Edited by Nooruddin Thajuddin*

Fungicides are a class of pesticides used for killing or inhibiting the growth of fungus. They are extensively used in pharmaceutical industry, agriculture, in protection of seed during storage and in preventing the growth of fungi that produce toxins. Hence, fungicides production is constantly increasing as a result of their great importance to agriculture. Some fungicides affect humans and beneficial microorganisms including insects, birds and fish thus public concern about their effects is increasing day by day. In order to enrich the knowledge on beneficial and adverse effects of fungicides this book encompasses various aspects of the fungicides including fungicide resistance, mode of action, management fungal pathogens and defense mechanisms, ill effects of fungicides interfering the endocrine system, combined application of various fungicides and the need of GRAS (generally recognized as safe) fungicides. This volume will be useful source of information on fungicides for post graduate students, researchers, agriculturists, environmentalists and decision makers.

Fungicides - Beneficial and Harmful Aspects

Fungicides

Beneficial and Harmful Aspects

*Edited by Nooruddin Thajuddin*

Photo by i-Stockr / iStock