*2.1.7. Phoma black stem (Phoma macdonaldii Boerema)*

*2.1.6. Charcoal rot [Macrophomina phaseolina (Tassi) Goild]*

608 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

of Italy suggests good adaptation of *Macrophomina* to the host.

description of charcoal rot has been provided by Aćimović [120].

Mihaljčević [122] adapted it for sunflower testing.

and *Rhizoctonia bataticola* (Taub.) Butler.

in most sunflower-growing countries.

for resistance to this pathogen.

virulent in affecting the head weight.

Synonyms for this fungus are *Sclerotium bataticola* Taub., *Macrophomina phaseoli* (Maubl.) Ashby

Charcoal rot causes economic damage to sunflower production in arid regions. It is widespread

Charcoal rot may cause premature death of sunflowers grown on light, sandy soil under hot

Manici *et al.* [117] concluded that the great variability in pathogenicity in all the climatic areas

This pathogen has been studied by many authors. Iliescu [118] and Ionita and Iliescu [119] published a detailed review of charcoal rot symptomatology, taxonomy, epidemiology, pathogenesis, and control of *Macrophomina* in sunflowers. To our knowledge, a most detailed

Walcz and Piszkev [121] have developed an inoculation method for screening sunflower lines

Mihaljčević [122, 123] conducted the most detailed studies on the effectiveness of inoculation methods with *Macrophomina*. According to his results, the method of Hsi (1961) was the best of the four inoculation methods tested. Hsi developed this method for sorghum testing and

Ahmad *et al.* [124] examined 13 exotic sunflower inbred lines and eight *Macrophomina* isolates. The tested inbred lines differed significantly in agronomic characteristics (head diameter, head weight, number of seeds per head, 1000-seed weight, and yield per unit area). The inbred lines HAR 1 and HAR 2 were resistant/tolerant across all charcoal rot isolates, while HA 822 was susceptible to the disease development and two charcoal rot isolates (MP9 and MP21) were

Mihaljčević [122] also found high resistance levels in lines derived from the Argentine cultivars Pehuan INTA, Ciro, and Klein as well as in the lines GVP-1 and GVP-2, derived from varietal populations (VNIIMK, Krasnodar) developed by interspecific hybridization with *H. tuberosus*.

Galina Pustovoit and Gubin [83] found the sources of resistance to *Macrophomina* in the F14 progenies of the interspecific hybrid VNIIMK8931 × *H. tuberosus*. A radical inoculation method (injecting fungus suspension into the head tissue) confirmed a complete resistance in 62 lines.

Studies of wild sunflower species have been insufficient to enable the identification of resistance genes as the sources of resistance against charcoal rot. Seiler [49] concluded that interspecific hybrids based on *H. tuberosus* have resistance to charcoal rot. Wild species *H. mollis*, *H. maximiliani*, *H. resinosus*, *H. tuberosus*, and *H. pauciflorus* have also shown resistance.

and dry climate. The disease is well known in the southern part of Europe [52].

According to Aćimović [120], the synonym for this fungus is *Phoma oleracea* var. *helianthituberosi* Sacc.

Phoma black stem is in large expansion in several countries in the world. It causes premature drying of plants (forced ripening) resulting in economic damage that increases from 1 year to another [22].

Viranyi [52] points out that *Phoma* black stem is extremely severe in France where basal stem lesions often result in lodging.

The inoculation method described by Maširević [125] is recommended to sunflower breeders. For efficiency, molecular markers should be used when screening breeding material.

Fayzalla [126] examined in detail the resistance to *Phoma macdonaldii* in a large set of Novi Sad genotypes of cultivated sunflower and several wild species. Using an inoculation method, he found that there was no satisfactory tolerance to *Phoma macdonaldii* in the genotypes of the cultivated sunflower. Among the wild species, however, high tolerance was registered in *H. maximiliani, H. argophillus, H. tuberosus,* and *H. pauciflorus.*

*Phoma* black stem resistance has been reported in several perennial species: *H. eggertii, H. hirsutus, H. resinosus,* and *H. tuberosus* [99].

Encheva *et al.*[110] stated that interspecies of hybrids based on *H. salicifolius* are highly resistant to *Phoma black stem.*

Christov [78] also confirms that interspecies hybrids based on *H. eggertii, H. debilis, and H. argophillus* exhibit *high* levels of *resistance* to *Phoma*.

Darwishzadeh *et al.* [127] undertook experiments to determine the partial resistance of sunflower genotypes to seven isolates and highly significant differences were observed among genotypes, isolates, and their interactions. Two genotypes exhibited specific resistance with a wide range of isolate-nonspecific partial resistance appearing as well. In addition, QTLs were also found associated with isolate-specific and nonspecific resistance [128]. Alignan *et al.* [129] developed a 1000-element cDNA microarray-containing genes putatively involved in primary metabolic pathways in order to identify genes responsible for partial resistance. They were successful in identifying 38 genes differently expressed among genotypes, treatments, and times.

According to Škorić [99], resistance to *Phoma* black stem is positively correlated with resistance to *Phomopsis* stem canker and charcoal rot.

#### *2.1.8. Alternaria blight (Alternaria helianthi Tub. et Nish.)*

Aćimović [120] cited the following synonyms for *Alternaria* blight: *Helminthosporium helianthi* Hansf., *Alternaria leucanthemum* Nelena et Vas. and *Embellisia helianthi* (Hansf.). The same author stated that sunflowers are also attacked by *Alternaria zinniae* Pape, *Alternaria alternata* (Fr.) Keiss (synonym *Alternaria tenuis* Ness.) and *Alternaria helianthinficiens* Simmons, Walcz, and Roberts. Of these species, *Alternaria helianthi* is the most common on sunflowers and the best studied from the point of view of sunflower resistance. It was found to attack sunflowers in all continents where this oilseed crop is grown. In the previous decade, it caused the most extensive economic damage on sunflowers in India and Brazil. According to Aćimović [120], most of the cultivated sunflower genotypes are sensitive to *Alternaria* blight.

Regina *et al.* [130] concluded that the occurrence of *Alternaria helianthi* in southern Brazil depended on the pathogen race and sunflower cultivar to a large extent. Attack is most intensive on crops sown in December and least intensive in October-sown crops. Dudienas *et al.* [131] claimed that *Alternaria* causes economic damage in Brazil, especially in humid conditions.

Aćimović [132] tested 1389 inbred lines for 4 years under field conditions and found that only six lines possessed satisfactory tolerance to *Alternaria* blight.

Madhavi *et al.* [133] found the sources of resistance to *Alternaria* blight in *H. tuberosus* and *H. occidentalis.*

Lipps and Herr [134] examined 496 sunflower genotypes for resistance to *Alternaria* for 3 years and found tolerance in eight genotypes only. A different situation was encountered when the *H. tuberosus* population was inoculated in the greenhouse. Based on the obtained results, the authors concluded that *H. tuberosus* can be used as a source of resistance to *Alternaria helianthi.*

Morris *et al.* [135] confirmed that all 21 annual taxa and 18 of 21 perennial species evaluated were susceptible to *A. helianthi* using applied spore suspensions, while perennial species *H. hirsutus, H. pauciflorus* ssp. *subrhomboideus,* and *H. tuberosus* appear to resist infection by *A. helianthi.*

Sujatha *et al.* [136] determined that nine perennial *Helianthus* species, *H. maximiliani, H. mollis, H. divaricatus, H. simulans, H. occidentalis, H. pauciflorus* and *H. decapetalus, H. resinosus*, and *H. tuberosus* were highly resistant to *Alternaria* leaf spot; all annuals were susceptible.

Christov [78] reported that perennial *H. decapetalus, H. laevigatus, H. glaucophyllus,* and *H. ciliaris* were potential sources of genes for *Alternaria* resistance.

Complete resistance to *Alternaria* leaf spot was reported in interspecific hybrids of *H. salicifo‐ lius* by Encheva *et al.* [110]. Škorić [81] obtained similar results.

#### **2.2. Other fungal diseases**

There is a large number of other fungal diseases of sunflower that cause economic damage to sunflower production in some regions and in some years. Unfortunately, most of them have not been included in breeding programs yet [22].

#### *2.2.1. Fusarium wilt (Fusarium spp.)*

According to Aćimović [120], several species of the genus Fusarium attack sunflowers: *Fusarium solani, Fusarium solani* var. *minus, Fusarium oxysporum, Fusarium oxysporum, F.* *helianthi, Fusarium moniliforme* (syn. *Gibberella fujikuroi*), *Fusarium equiseti, Fusarium tabacum, Fusarium culmorum, Fusarium* sp. and *Fusarium* spp.

Viranyi [52] states that *Fusarium wilt (Fusarium* spp.) has been reported as a pathogen of concern only from Russia [137] where it appeared to be harmful for sunflower production. Based on the extent of necrosis incited by the fungus on the main root and the root–hypocotyl transition zone of sunflower seedlings, some tolerance to pathogen attack could be detected among the genotypes [138]. In a breeding program, a number of new breeding lines were developed exhibiting relatively good field tolerance [139].

There are few research papers dealing with sunflower resistance to *Fusarium*. In one of these earlier papers, Orellana [140] reported that out of 49 inbred lines tested, 23 were resistant to *Fusarium moniliforme.* In recent years, Goncharov [139] produced plants tolerant to *Fusarium* on the basis of laboratory tests and individual selection of plants from three double-cross combinations and an F3 cross (UV.680 × o.p. *cv.* Leader).

### *2.2.2. Rhizopus head rot (Rhizopus spp.)*

and Roberts. Of these species, *Alternaria helianthi* is the most common on sunflowers and the best studied from the point of view of sunflower resistance. It was found to attack sunflowers in all continents where this oilseed crop is grown. In the previous decade, it caused the most extensive economic damage on sunflowers in India and Brazil. According to Aćimović [120],

Regina *et al.* [130] concluded that the occurrence of *Alternaria helianthi* in southern Brazil depended on the pathogen race and sunflower cultivar to a large extent. Attack is most intensive on crops sown in December and least intensive in October-sown crops. Dudienas *et al.* [131] claimed that *Alternaria* causes economic damage in Brazil, especially in humid

Aćimović [132] tested 1389 inbred lines for 4 years under field conditions and found that only

Madhavi *et al.* [133] found the sources of resistance to *Alternaria* blight in *H. tuberosus* and *H.*

Lipps and Herr [134] examined 496 sunflower genotypes for resistance to *Alternaria* for 3 years and found tolerance in eight genotypes only. A different situation was encountered when the *H. tuberosus* population was inoculated in the greenhouse. Based on the obtained results, the authors concluded that *H. tuberosus* can be used as a source of resistance to *Alternaria helianthi.*

Morris *et al.* [135] confirmed that all 21 annual taxa and 18 of 21 perennial species evaluated were susceptible to *A. helianthi* using applied spore suspensions, while perennial species *H. hirsutus, H. pauciflorus* ssp. *subrhomboideus,* and *H. tuberosus* appear to resist infection by *A.*

Sujatha *et al.* [136] determined that nine perennial *Helianthus* species, *H. maximiliani, H. mollis, H. divaricatus, H. simulans, H. occidentalis, H. pauciflorus* and *H. decapetalus, H. resinosus*, and *H.*

Christov [78] reported that perennial *H. decapetalus, H. laevigatus, H. glaucophyllus,* and *H.*

Complete resistance to *Alternaria* leaf spot was reported in interspecific hybrids of *H. salicifo‐*

There is a large number of other fungal diseases of sunflower that cause economic damage to sunflower production in some regions and in some years. Unfortunately, most of them have

According to Aćimović [120], several species of the genus Fusarium attack sunflowers: *Fusarium solani, Fusarium solani* var. *minus, Fusarium oxysporum, Fusarium oxysporum, F.*

*tuberosus* were highly resistant to *Alternaria* leaf spot; all annuals were susceptible.

*ciliaris* were potential sources of genes for *Alternaria* resistance.

*lius* by Encheva *et al.* [110]. Škorić [81] obtained similar results.

not been included in breeding programs yet [22].

most of the cultivated sunflower genotypes are sensitive to *Alternaria* blight.

six lines possessed satisfactory tolerance to *Alternaria* blight.

610 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

conditions.

*occidentalis.*

*helianthi.*

**2.2. Other fungal diseases**

*2.2.1. Fusarium wilt (Fusarium spp.)*

Dry rot of sunflower is caused by the following fungi from the genus *Rhizopus: Rhizopus arrhizus* Fisch. (syn. *Rhizopus nodosus* Namysl.), *Rhizopus nigricans* Ehr. (syn. *Rhizopus stoloni‐ fer* Eh. et Fr.) and *Rhizopus oryzae* Nent et Geer [120].

Dry rot occurs typically in regions with dry climate and high temperatures. It often causes a significant yield reduction and it particularly reduces the oil content in seeds [22].

It has become an important disease of sunflower in the USA [141]. The disease reduced oil quality and quantity in oilseed sunflower [142]. Infection of sunflower with *Rhizopus* head rot is enhanced by larval feeding of sunflower moth, *Homoeosoma electellum* (Hulst), which contributes to a secondary infection [141].

Yang *et al.* [143] reported that 4 out of 32 wild species and subspecies were resistant when inoculated with *R. arrhizus* and *R. oryzae* Went. The resistant sources were perennial *H. divaricatus, H. hirsutus, H. xlaetiflorus,* and *H. resinosus.*

One of the pioneer works was that of Agrawat *et al.*[144], who studied the resistance to *Rhizopus nodosus* in 91 sunflower cultivars. Their results based on an inoculation test indicated that resistance existed only in cultivars – Armavirec, Armavirskiy 3497, EC 40277, and K-2217, all from Krasnodar.

Rhizopus head rot brings great economic damage in many countries, by decreasing seed yield, seed oil content, and seed development. Unfortunately, few researchers in the world work on the examination of this pathogen.

#### *2.2.3. Powdery mildew (Erysiphe cichoracearum DC)*

Sunflower is a host to three fungal genera that cause powdery mildew [120, 145]: *Erysiphe cichoracearum* DC, which is widespread in all continents where sunflower are grown, *Leveillula compositarum* Golow., *Leveillula taurica* (Lev.) Arn., and *Sphaerotheca fuliginea* (Schlecht. ex Fr.) Poll.

Since *Erysiphe cichoracearum* DC is common on sunflowers around the world, resistance to this pathogen has been studied most extensively. Saliman *et al.* [146] was among the first to identify wild species from the genus *Helianthus* resistant to *Erysiphe* [120].

Jan and Chandler [147] transferred the resistance from *H. debilis* Nutt. into the line P21. The mode of inheritance in this resistance source was partially dominant. According to unpub‐ lished results of Škorić, resistance to powdery mildew exists in several inbred lines, especially those that incorporate genes from *H. tuberosus.*

Breeding programs conducted in Argentina, Australia, and the Republic of South Africa have been targeted on Albugo resistance and several highly tolerant hybrids were obtained.

Seiler [49] indicates that *Helianthus debilis* ssp. *praecox*, and *H. bolanderi,* and 14 perennial species were tolerant of powdery mildew in both field and greenhouse tests [146]. Not all population of perennial species are resistant: population of *H. grosseserratus* and *H. maximiliani* showed differential reactions. Škorić [116] reported that interspecific hybrids with *H. giganteus, H. hirsutus, H. divaricatus,* and *H. salicifolius* had no powdery mildew symptoms.

Jan and Chandler [147] transferred the resistance from *H. debilis* Nutt. into the line P21. The mode of inheritance in this resistance source was partially dominant. According to unpub‐ lished results of Škorić, resistance to powdery mildew exists in several inbred lines, especially those that incorporate genes from *H. tuberosus.*

#### *2.2.4. Botrytis cinerea Pers.*

Sunflower geneticists and breeders have unjustly neglected the polyphagous fungus *Botrytis cinerea* Pers., although it causes economic damage in sunflower production in some regions.

Prats [148] was the first to discover a source of resistance to *Botrytis cinerea* in the cultivar INRA 64-01.

Burlov and Artemenko [149] found the line Od-2624 to be resistant to *Botrytis.*

Kostyuk [150] studied some 1400 sunflower genotypes and found that none of them were resistant and only some were tolerant to *Botrytis* under natural and inoculation conditions.

#### *2.2.5. White rust (Albugo tragopogonis Schr.)*

According to Aćimović [120] the synonym for this fungus is *Albugo tragopogonis* (Pers.) Schr. White rust has been registered on sunflowers in several countries and is particularly aggressive in South America (Argentina), Africa (Republic of South Africa), some Asian countries, several countries from the former Soviet Union, and Australia.

Breeding programs conducted in Argentina, Australia, and the Republic of South Africa have been targeted on *Albugo* resistance and several highly tolerant hybrids were obtained [22].

An established breeding centre, which focuses its research on identifying sources of resistance to white rust in wild species of genus *Helianthus*, unfortunately does not exist anywhere in the world.

### **2.3. Viruses and Bacteria**

Since *Erysiphe cichoracearum* DC is common on sunflowers around the world, resistance to this pathogen has been studied most extensively. Saliman *et al.* [146] was among the first to identify

Jan and Chandler [147] transferred the resistance from *H. debilis* Nutt. into the line P21. The mode of inheritance in this resistance source was partially dominant. According to unpub‐ lished results of Škorić, resistance to powdery mildew exists in several inbred lines, especially

Breeding programs conducted in Argentina, Australia, and the Republic of South Africa have been targeted on Albugo resistance and several highly tolerant hybrids were obtained.

Seiler [49] indicates that *Helianthus debilis* ssp. *praecox*, and *H. bolanderi,* and 14 perennial species were tolerant of powdery mildew in both field and greenhouse tests [146]. Not all population of perennial species are resistant: population of *H. grosseserratus* and *H. maximiliani* showed differential reactions. Škorić [116] reported that interspecific hybrids with *H. giganteus, H.*

Jan and Chandler [147] transferred the resistance from *H. debilis* Nutt. into the line P21. The mode of inheritance in this resistance source was partially dominant. According to unpub‐ lished results of Škorić, resistance to powdery mildew exists in several inbred lines, especially

Sunflower geneticists and breeders have unjustly neglected the polyphagous fungus *Botrytis cinerea* Pers., although it causes economic damage in sunflower production in some regions.

Prats [148] was the first to discover a source of resistance to *Botrytis cinerea* in the cultivar INRA

Kostyuk [150] studied some 1400 sunflower genotypes and found that none of them were resistant and only some were tolerant to *Botrytis* under natural and inoculation conditions.

According to Aćimović [120] the synonym for this fungus is *Albugo tragopogonis* (Pers.) Schr. White rust has been registered on sunflowers in several countries and is particularly aggressive in South America (Argentina), Africa (Republic of South Africa), some Asian countries, several

Breeding programs conducted in Argentina, Australia, and the Republic of South Africa have been targeted on *Albugo* resistance and several highly tolerant hybrids were obtained [22].

An established breeding centre, which focuses its research on identifying sources of resistance to white rust in wild species of genus *Helianthus*, unfortunately does not exist anywhere in the

*hirsutus, H. divaricatus,* and *H. salicifolius* had no powdery mildew symptoms.

Burlov and Artemenko [149] found the line Od-2624 to be resistant to *Botrytis.*

wild species from the genus *Helianthus* resistant to *Erysiphe* [120].

612 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

those that incorporate genes from *H. tuberosus.*

those that incorporate genes from *H. tuberosus.*

*2.2.5. White rust (Albugo tragopogonis Schr.)*

countries from the former Soviet Union, and Australia.

*2.2.4. Botrytis cinerea Pers.*

64-01.

world.

Some viruses are capable of causing disease in sunflowers. The number of viruses that are specific and attack only sunflowers is very limited. In most cases, the main host is another agricultural crop and sunflower is only a secondary host [120]. According to Gulya *et al.* [145], several viruses attack sunflower: aster yellows virus, cucumber mosaic virus, sunflower mosaic virus, sunflower ringspot virus, sunflower yellow blotch virus, leaf crinkle virus, tobacco ringspot virus, tobacco streak virus, tomato spotted wilt virus, potyvirus, etc.

Viruses are typically transmitted by vectors, the most important among which are aphids.

Srechari *et al.* [151] reported three aphid species, *Aphis gossypii* Glove., *Aphis craccivora* Koch, and *Rhopalosiphum maidis* (Fitch), as virus vectors. Among them, *A. gossypii* is best known as the vector that transmits the sunflower mosaic disease.

Lenardon *et al.*[152] detected the sunflower chlorotic mottle virus (SuCMoV) in several regions of Argentina.

Lenardon *et al.* [153] tested 232 lines in the greenhouse using an inoculation method. Only three lines exhibited partial resistance (L33, L74, and L42) to the sunflower chlorotic mottle virus. Of these three lines, L33 was the most resistant. Screening F2 population of crosses between resistant and sensitive lines in the greenhouse and in field, the authors concluded that the resistance is controlled by a single dominant gene (Remo-1).

In recent years, the sunflower chlorotic mottle virus has been studied intensively at the molecular level.

Dujovny *et al.* [153] conducted a molecular characterization of a new potyvirus (SuCMoV). Arias *et al.* [155] described the effect of SuCMoV on some aspects of carbon metabolism in sunflower plants.

Mailo *et al.* [156] mechanically inoculated one sensitive (20 016) and one tolerant line (B-133) with SuCMoV. Total RNA was isolated from infected leaf tissue for study at the molecular level. The achieved results indicated that the gene expression profiles in the inoculated plants (of the sensitive and the tolerant line) were statistically significant compared with leaves of plants that were not inoculated. Eighty-eight genes were differentially expressed in the tolerant line.

#### *2.3.1. Bacterial diseases*

Bacterial diseases of sunflower are caused by pathogenic bacteria. They can be found on sunflowers in most countries where this oil crop is grown. In addition to sunflower, most of these bacteria also attack other crops. The sunflower is typically a secondary host and quite rarely the main host [120].

The most widespread bacteria on sunflowers are *Agrobacterium tumefaciens* (E. F. Sm. and Town.) Conn, *Pseudomonas syringae* pv. *tabaci* (Wolf and Foster 1917) Young, Dye and Wilkie 1978 (synonyms *Pseudomonas tabaci* and *Bacterium tabacum* Wolf and Foster), *Xanthomonas campestris* pv. *phaseoli* (Smith) Due., *Pseudomonas syringae* pv. *helianthi* (Kawamura) Dye, Wilkie and Young, *Pseudomonas solanacearum* (Smith), *Erwinia carotovora* pv. *carotovora* (Jones) Bergey *et al.*, etc. [120].

There are few papers in the literature dealing with sunflower selection for resistance to bacterial diseases.

Among these few, Nemeth and Walcz [157] reported the occurrence of *Erwinia carotovora* on sunflowers in Hungary in the period 1984–1986. Testes of inbred lines and commercial hybrids conducted under natural conditions indicated that there existed significant differences in resistance to this bacterial disease. However, the tests showed that the breeding material can be tested by inoculation methods under field conditions.

#### **2.4. Sunflower breeding for resistance to broomrape (***Orobanche cumana* **Wallr.)**

The parasitic angiosperm broomrape (*Orobanche cumana* Wallr. = *Orobanche cernua* Loelf.) is the cause of many economic losses in sunflower production in a number of countries in the world, especially in central and eastern Europe, Spain, Turkey, Israel, Kazakhstan, and China. Its presence has also been established in Australia.

Sunflower breeders have been fighting *Orobanche cumana* Wallr. for almost a century [22].

According to past researches, there have been different mechanisms of sunflower resistance to *Orobanche*. Most often these are genetic mechanisms, but there are also physiological, biochemical, mechanical, and others.

According to Morozov [158], the first reports of broomrape in sunflower came from Saratov Oblast in Russia and date back to the 1890s. The same author mentions that the first sunflower varieties resistant to race A of *Orobanche* were developed by Plachek at the Saratov breeding station.

At the beginning of the 20th century, broomrape spread across Russia significantly and endangered the mass production of sunflower. The first cultivar resistant to race A, Saratovskij 169, was created by Plachek. In the years that followed, other cultivars resistant to race A were also produced (Kruglik A/41, Zelenka, and Fuksinka). As the mass production of sunflower spread quickly, it was followed by a relatively fast production of a new race called B. Zhdanov in Rostov on the Don announced that he had produced several cultivars resistant to a new race (B). During the period 1924–1960, Pustovoit in VNIIMK, Krasnodar created highly productive cultivars, which were resistant to race B [22, 158].

In order to attain their breeding goals and identify sources of broomrape resistance, sunflower breeders must develop a breeding strategy, decide on a breeding method, secure the necessary germplasm and differential lines for broomrape race identification, and choose the appropriate inoculation method and molecular marker technique (marker-assisted selection (MAS)) – Škorić [22].

Vrânceanu *et al.* [159] defined a set of differential lines for the evaluation of the composition of broomrape races. Among them was the AD-66 line, which represented a tester line suscep‐ tible to all broomrape races. On the other hand, the differential line (cultivar) Kruglik A41 was used for race A, the Jdanov-8281 cultivar for race B, the Romanian cultivar Record for race C, line S-1348 for race D, and line P-1380-2 for race E. Pâcureanu-Joița *et al.* [160] included the line LC-1093 as a tester for race F. Unfortunately, there have been no tester lines for the latest broomrape races [160].

and Young, *Pseudomonas solanacearum* (Smith), *Erwinia carotovora* pv. *carotovora* (Jones) Bergey

There are few papers in the literature dealing with sunflower selection for resistance to

Among these few, Nemeth and Walcz [157] reported the occurrence of *Erwinia carotovora* on sunflowers in Hungary in the period 1984–1986. Testes of inbred lines and commercial hybrids conducted under natural conditions indicated that there existed significant differences in resistance to this bacterial disease. However, the tests showed that the breeding material can

The parasitic angiosperm broomrape (*Orobanche cumana* Wallr. = *Orobanche cernua* Loelf.) is the cause of many economic losses in sunflower production in a number of countries in the world, especially in central and eastern Europe, Spain, Turkey, Israel, Kazakhstan, and China.

Sunflower breeders have been fighting *Orobanche cumana* Wallr. for almost a century [22].

According to past researches, there have been different mechanisms of sunflower resistance to *Orobanche*. Most often these are genetic mechanisms, but there are also physiological,

According to Morozov [158], the first reports of broomrape in sunflower came from Saratov Oblast in Russia and date back to the 1890s. The same author mentions that the first sunflower varieties resistant to race A of *Orobanche* were developed by Plachek at the Saratov breeding

At the beginning of the 20th century, broomrape spread across Russia significantly and endangered the mass production of sunflower. The first cultivar resistant to race A, Saratovskij 169, was created by Plachek. In the years that followed, other cultivars resistant to race A were also produced (Kruglik A/41, Zelenka, and Fuksinka). As the mass production of sunflower spread quickly, it was followed by a relatively fast production of a new race called B. Zhdanov in Rostov on the Don announced that he had produced several cultivars resistant to a new race (B). During the period 1924–1960, Pustovoit in VNIIMK, Krasnodar created highly productive

In order to attain their breeding goals and identify sources of broomrape resistance, sunflower breeders must develop a breeding strategy, decide on a breeding method, secure the necessary germplasm and differential lines for broomrape race identification, and choose the appropriate inoculation method and molecular marker technique (marker-assisted selection (MAS)) –

Vrânceanu *et al.* [159] defined a set of differential lines for the evaluation of the composition of broomrape races. Among them was the AD-66 line, which represented a tester line suscep‐ tible to all broomrape races. On the other hand, the differential line (cultivar) Kruglik A41 was

**2.4. Sunflower breeding for resistance to broomrape (***Orobanche cumana* **Wallr.)**

be tested by inoculation methods under field conditions.

614 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

Its presence has also been established in Australia.

cultivars, which were resistant to race B [22, 158].

biochemical, mechanical, and others.

station.

Škorić [22].

*et al.*, etc. [120].

bacterial diseases.

Genes for resistance to broomrape races A, B, C, and D are present in varietal populations of sunflower developed in breeding programs from Krasnodar, Armavir, Odessa, Fundu‐ lea, and several other places [149]. Genes that confer resistance to races E, F, G, and the latest ones, on the other hand, have been identified in certain wild species of the genus *Helian‐ thus* and have been incorporated into cultivated sunflower genotypes by interspecific hybridization [161, 162].

Galina Pustovoit [163] and her team made a great contribution in this area by developing sunflower varieties through interspecific hybridization in which *H. tuberosus* was used as the donor of *Or* genes. These varieties were used in the identification of *Or*4 and *Or*6 genes.

Fernandez-Martinez *et al.* [164] tested 44 wild sunflower accessions (representing 27 perennial and 4 annual species) and 44 cultivated sunflower accessions, which they raised in a growth chamber and then transplanted to a greenhouse. The material was inoculated with the virulent race F (population SE 296). Most of the perennial species proved fully resistant to race F.

Among the wild annual species, *H. anomalus* and *H. agrestis* were completely resistant, while *H. debilis* ssp. *cucumerifolius* and *H. exilis* segregated with regard to *Orobanche* resistance [164].

Interspecific hybrids based on *H. eggertii* and *H. smithii* showed total resistance to broomrape in Bulgaria [107]. Broomrape resistance to the local race in Bulgaria was reported in *H. divaricatus*, *H. eggertii*, *H. giganteus*, *H. grosseserratus*, *H. glaucophyllus*, *H. mollis*, *H. nuttallii*, *H. pauciflorus* (=*rigidus*), *H. resinosus*, and *H. tuberosus* [107, 165].

Diploid perennial species *H. divaricatus*, *H. giganteus*, *H. glaucophyllus*, *H. grosseserratus*, *H. mollis*, *H. nuttallii*, and *H. smithii* and their interspecific hybrids were resistant to broomrape [67]. Christov [78] reported that several perennial *Helianthus* species showed 100% resistance including *H. tuberosus*, *H. eggertii*, *H. smithii*, *H. pauciflorus*, and *H. strumosus*.

Jan *et al.* [166] crossed the wild sunflower species *H. maximiliani* Schrad, *H. grosseserratus* Mart., and *H. divaricatus* L. with cultivated sunflower and developed four populations (BR1-BR4) resistant to race F in Spain.

Numerous authors in public institutions and private companies use wild sunflower species as donors of genes for resistance to broomrape.

The sources of resistance to broomrape, which have been discovered so far, mostly use the gene of resistance taken from the wild species of the genus *Helianthus*. According to the results obtained so far, there are over 20 wild species of the genus *Helianthus*, which contain the gene of resistance to broomrape [22].

When broomrape occurred, breeders used infested fields for testing selection materials for broomrape resistance in many countries. This method is not reliable enough since in natural conditions infestation with broomrape on those fields is not equally spread, which causes large experimental errors. A much more reliable method, which is applied on testing resistance, is mixing of broomrape seed (a certain amount) with the selection material in the process of cultivation. In order to accelerate the process of testing the resistance of selection material, there are certain containers used in a greenhouse during the period autumn/winter, which are filled with a mixture of soil and sand filled with broomrape seeds and the seed of sunflower genotypes that are being tested [22].

Panchenko [167] developed a method of testing the selection materials in the greenhouse, which enables simultaneous testing of a great number of lines that are being created. The purpose of this method is preparation of a medium (sterilized soil + sand or perlite) on the tables in the medium. Following that, the appropriate amount of broomrape seed is added and the selection material is cultivated. Within 3–4 weeks after germination, it is possible to make the evaluation of resistance.

In Rustica Prograin Genetique, Grezes-Besset [168] developed a fast method of testing the selection material of sunflower in plastic tubes (a mixture of sand and perlite), which enables a reliable testing of a large number of lines (hybrids) in small space and the cycle lasts about 3 weeks.

However, the most reliable and the most easily applied method of screening breeding materials for broomrape resistance is the use of molecular markers, QTL, RFLP, RAPD, TRAOP, and SSR markers which have been used for this purpose.

Increased use of marker-assisted selection, which gives quick and reliable results, is very positive for sunflower breeding.

The best example of that is the production of hybrids resistant to the imidazolinone group of herbicides, which has proven itself in mass production by cultivating IMI-resistant hybrids followed by controlling broomrape. IMI-resistant hybrids are very important in regions where new races of broomrape have occurred [22].

Dominant genes for resistance to races A, B, C, D, E, and F have been found and incorporated into cultivated sunflower genotypes. In the last 2–3 years, new broomrape populations have been discovered in several countries. None of the existing commercial hybrids resistant to races A, B, C, D, E, and F have proven resistant to these new populations.

#### **2.5. Sunflower breeding for insect resistance**

Several hundred different species of insects cause infestations in sunflower. However, economic losses are caused only by a few insect species [169]. Some insects transmit several sunflower diseases [170]. *Homoeosoma* spp. are a significant problem in cultivated sunflower on four continents. *Homoeosoma nebulella* (Hubner) infests sunflower in Europe and Asia. In South America, sunflower is infested by *H. heinrichi* (Pastrana), whereas *H. electellum* (Hulst) causes damage to sunflower in Mexico, USA, and Canada.

Cultivar resistance to European sunflower moth (*H. nebulella*) was incorporated in USSR 60-70 years ago through interspecies hybridization of cultivated sunflower and *H. tuberosus* spp. *Purpurellus,* Cockerell [171, 172].

The mechanism of resistance to sunflower moth exists due to the phytomelanin (carbon) layer in the husk. Black hull colour is positively correlated with phytomelanin content in the husk.

experimental errors. A much more reliable method, which is applied on testing resistance, is mixing of broomrape seed (a certain amount) with the selection material in the process of cultivation. In order to accelerate the process of testing the resistance of selection material, there are certain containers used in a greenhouse during the period autumn/winter, which are filled with a mixture of soil and sand filled with broomrape seeds and the seed of sunflower

Panchenko [167] developed a method of testing the selection materials in the greenhouse, which enables simultaneous testing of a great number of lines that are being created. The purpose of this method is preparation of a medium (sterilized soil + sand or perlite) on the tables in the medium. Following that, the appropriate amount of broomrape seed is added and the selection material is cultivated. Within 3–4 weeks after germination, it is possible to make

In Rustica Prograin Genetique, Grezes-Besset [168] developed a fast method of testing the selection material of sunflower in plastic tubes (a mixture of sand and perlite), which enables a reliable testing of a large number of lines (hybrids) in small space and the cycle lasts about

However, the most reliable and the most easily applied method of screening breeding materials for broomrape resistance is the use of molecular markers, QTL, RFLP, RAPD, TRAOP, and

Increased use of marker-assisted selection, which gives quick and reliable results, is very

The best example of that is the production of hybrids resistant to the imidazolinone group of herbicides, which has proven itself in mass production by cultivating IMI-resistant hybrids followed by controlling broomrape. IMI-resistant hybrids are very important in regions where

Dominant genes for resistance to races A, B, C, D, E, and F have been found and incorporated into cultivated sunflower genotypes. In the last 2–3 years, new broomrape populations have been discovered in several countries. None of the existing commercial hybrids resistant to races

Several hundred different species of insects cause infestations in sunflower. However, economic losses are caused only by a few insect species [169]. Some insects transmit several sunflower diseases [170]. *Homoeosoma* spp. are a significant problem in cultivated sunflower on four continents. *Homoeosoma nebulella* (Hubner) infests sunflower in Europe and Asia. In South America, sunflower is infested by *H. heinrichi* (Pastrana), whereas *H. electellum* (Hulst)

Cultivar resistance to European sunflower moth (*H. nebulella*) was incorporated in USSR 60-70 years ago through interspecies hybridization of cultivated sunflower and *H. tuberosus* spp.

A, B, C, D, E, and F have proven resistant to these new populations.

causes damage to sunflower in Mexico, USA, and Canada.

genotypes that are being tested [22].

616 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

SSR markers which have been used for this purpose.

the evaluation of resistance.

positive for sunflower breeding.

new races of broomrape have occurred [22].

**2.5. Sunflower breeding for insect resistance**

*Purpurellus,* Cockerell [171, 172].

3 weeks.

North-American species of sunflower moth (*H. electellum*) is far more virulent on cultivated sunflower than *H. nebulella*; hence, resistance breeding to this insect is of great importance in North America.

According to the results of Rogers and Kreitner [173], the presence of phytomelanin in sunflower seed pericarp prevents seed infestation with *H. electellum*. By monitoring the formation of pericarp (husk), it was determined that phytomelanin starts to accumulate between the hypodermis and sclerenchyma 3 days after fertilization, whereas its formation is clearly visible 13 days after fertilization.

In the major production area of North America, there are about 14 principal insect pests of cultivated sunflower, and of this total about six are considered potential economic pests [174].

"Two breeding procedures are recommended for identifying lines with improved resistance to insects attacking cultivated sunflower. These procedures are based on the initial evidence that the resistance to the insects is quantitatively inherited, that is, controlled by several genes. Both are based on recurrent selection and random mating, with the main objective to combine as many of the alleles controlling resistance as possible" [175].

"The recurrent phenotypic selection breeding procedure could be utilized for selection against stem and/or foliage infesting insects. An original (C0) source population may be created by random mating cultivars or lines (e.g., Plant introductions, open-pollinated populations), which are then screened for resistance to a particular insect attack sunflower" [175].

"Recurrent phenotypic selection with S1 line progeny evaluation could be utilized for selection for head and/or seed infesting insects. An original (C0) source population is created similarly as in the recurrent phenotypic selection procedure. The C0 population is planted in a normal breeding nursery with the most vigorous plants selected for bagging and selfpollination" [175].

According to Seiler [49], the insects causing most economic damage in North America are: sunflower beetle [*Zygogramma exclamationis* (Fabritius)], the sunflower stem weevil [*Cylindro‐ copturus adspersus* (LeConte)], the red and gray seed weevils [*Smicronyx fulvus* (LeConte) and *S. sordidus* (LeConte)], the banded sunflower moth (*Cochylis hospes*Walsingham), the sunflower moth [*Homoeosoma electellum* (Hulst)], and the sunflower midge *Contarinia schulzi* Gagne. The sunflower head moth, *Homoeosoma electellum* is the most widespread and damaging sunflower insect pest in North America, while in Europe and Asia it is *Homoeosoma nebulella* (Hubner).

According to the results obtained by Rogers [176, 177], the following sunflower species exhibit significant levels of resistance to sunflower moth: *H. arizonensis*, *H. ciliaris, H. pumilus*, *H. resinosus*, *H. rigidus* × *laetiforus*, *H. silphiodes* and *H. smithii*.

Among the insects that cause economic losses to sunflower production, the biggest success was achieved in breeding for resistance to sunflower head moth above all in Europe by the development of cultivars in which an armored layer was induced in the husk from some specific wild species. Similar results were obtained in North America.

High level of resistance to *Bothynus gibbosus* was exhibited by the following species: *H. tuberoses, H. maximiliani, H. niveus, H. xlaetiflorus, H. salicifolius, H. mollis, H. grosseseratus, H. Argophyllus,* and *H. ciliar*is [178].

The results of Rogers [176, 179, 180], as well as Rogers and Thompson [178, 181, 182], confirm high levels of resistance to *Zygogramma exclamationis, Bothynus gibbosus, Masonaphis masoni* and *Empoasca abrupta* in wild sunflower species *H. tuberosus* and *H. maximiliani,* and recommend the use of these species in breeding programs.

Results of Rogers and Thompson [183, 184] confirm significant levels of resistance in two annual and 10 perennial wild species (*Masonaphis masoni*). The highest resistance to aphids was seen in *H. carnosus, H. exilis, H. floridanus* and *H. radula.*

Weak point in sunflower breeding for resistance to insects is that only few sunflower research‐ ers deal with this issue. Insecticides can be used to some extent, more or less successfully, as a solution to this problem in some species.

#### **2.6. Conclusions**

Biotic stresses cause great economic damages and act as a limiting factor for the production of sunflower.

Diseases are the main problem among biotic stresses. Using wild sunflower species of the *Helianthus* genus, genes conferring resistance to most dominant diseases were discovered and incorporated to the genotypes of the cultivated sunflower.

Regarding the achievements in sunflower breeding for disease resistance, the results can be divided into four different groups.

The first group consists of work that resulted in the discovery of genetic resistance to certain causative agents of sunflower diseases (*Plasmopara halstedii*, *Puccinia helianthi, Verticillium dahliae, Verticillium albo-atrum,* and *Erysiphe cichoracearum*).

The second group comprises work in which a high level of tolerance (field resistance) was achieved. This group includes the results achieved in breeding for resistance to *Phomopsis/ Diaporthe helianthi, Macrophomina phaseolina, Albugo tragopogonis,* and *Alternaria* ssp.

The third group consists of results in which a satisfactory level of tolerance was achieved (*Phoma macdonaldii* and to some extent *Sclerotinia sclerotiorum*).

The fourth group consists of results that were partly achieved, where the level of favorable tolerance, that is, resistance, was not reached (*Rhizopus* ssp., *Botrytis cinerea* and other fungal pathogens).

Viruses and bacteria only pose a minor problem in comparison with diseases. Breeding for resistance to viruses and bacteria also includes wild sunflower species.

Broomrape (*Orobanche cumana* Walr.) is a major global issue in sunflower production, partic‐ ularly in Central and Eastern Europe. Genes conferring resistance to six races of broomrape have been discovered in some wild sunflower species and incorporated into genotypes of the cultivated sunflower. Research work, which aims at finding genes of resistance to the newest races within specific wild sunflower species, is in progress.

Sources of resistance to imidazolines and sulfonylurea herbicides (tribenuron-methyl) have been found in a population of wild *H. annuus* from Kansas and incorporated in cultivated sunflower genotypes. Moreover, genes conferring resistance to these herbicides were discov‐ ered using induced mutations. The newly developed hybrids, resistant to the abovementioned herbicides, provide successful weed and broomrape control through imidazolines.

Insects are a major issue in sunflower production, especially in North America. Significant results have been obtained through breeding for resistance to sunflower head moth. Wild sunflower species are used in research work aimed at finding the sources of resistance to other economically harmful insects.

Different biotechnological methods (tissue culture, embryo culture, protoplast fusion, molec‐ ular markers, in vitro screening, and other methods) have been included in breeding for resistance to biotic stresses.
