**1. Introduction**

244 Fungicides for Plant and Animal Diseases

Zadoks, J. C., Chang, T. T. & Konzak, C. F. 1974. A decimal code for the growth stages of

#### **1.1** *In vitro* **culture**

*In vitro* plant culture which encompasses cell, tissue, organ and also embryo culture has been a vital technique for mass multiplication of plants (propagules), elimination of plant diseases thorough meristematic tissue culture technique, plant conservation (through cryopreservation) and crop improvement through gene transfer (Pierik, 1987; George, 1993; Singh & Chand, 2003; Sarasan et al., 2011). *In vitro* culture techniques have been used to shorten breeding cycles of plants and to achieve genetic transformation (Singh & Chand, 2003). Despite the several merits of *in vitro* culture techniques to the modern world where plant diseases, flower/fruit abortions and low plant regeneration capacity are major challenges, many developing countries, especially in Africa do not utilize them to address the numerous challenges in crop production and improvements. This is mainly due to the high investment costs in equipment, chemicals and intensive and skilled labor demands (Sarasan et al., 2011).

The high costs for equipment, water, chemicals (such as plant growth hormones or regulators, surface sterilants, disinfectants etc.) and culture losses due to *in vitro* culture contamination among many others can cripple several *in vitro* plant culture techniques in many developing countries, especially in Africa. Possibly, this is the main reason we have limited or no interest to invest in tissue culture laboratories in Africa, except for the academic institutions for the sole purpose of learning the technique.

#### **1.2** *In vitro* **contaminants**

The most common challenge for *in vitro* plant culture protocols in the tropics, especially stock plants derived from mature ortet has been elimination of culture contaminants. These culture contaminants have been problematic to the effect that there are instances where

Efficacy and Utilization of Fungicides and Other Antibiotics for Aseptic Plant Cultures 247

The fungi colonizing the plants could be endogenous, cryptic or endophytic. Herman (1990) reported that endogenous or endophytic microbes are often difficult to decontaminate. It is known that many endophytes are beneficial for the growth of ortet plants in the wild, but they also become contaminants in culture media. Helander et al. (1996) reported that mutualism between endophytes and the host plant depends on the prevailing plant condition. Endophytes can be harmful to plant cultures although they are useful outside the plant culture laboratories. Examples of some trees that live and thrive with symbiotic microbes (mycorrhizae) include *Uapaca kirkiana* (Mwamba, 1995), but this mutualistic

In the tropics, fungal contaminants are very common due to the prevailing favorable environmental conditions (especially the high temperatures and humidity) for the proliferation of fungal pathogens on the ortet plants. Furthermore, the conditions in the plant culture incubation chambers are favorable for the rapid growth of fungi and other *in vitro* contaminants. For instance, the range of temperatures (usually between 23 – 26 oC), high humidity and plant nutrients (available inside the culture vials or test-tubes) cannot deter or inhibit the proliferation of many fungi in the tubes placed in the culture incubation chambers. Moreover, some plants live and survive by mutual association with fungi in the

wild. However, such fungi are likely to become '*vitropathogenic*' in the plant cultures.

Generally, exogenous fungal pathogens are easy to eliminate from the stock plants using many fungicides. This is because such pathogens are found on the outer surface of the stock plants, and hence adequate contact between the fungicides and the pathogens and even the spores. However, insufficient surface disinfection might not dislodge exogenous pathogens and spores from the stock plants, and hence endogenous contaminants still pose a threat in

It is always difficult to detect the presence of either exogenous, endogenous or both pathogens on the stock plants. So it might be advisable to use systemic fungicides (e.g. Benomyl) before other disinfectants such as sodium hypochlorite are used. It is also a good practice to ensure that all possible culture contaminants are dealt with at the initial decontamination stage instead of re-disinfecting the explants at later stages (sub-culturing stages). Re-decontamination of explants might weaken the ensuing explants due to cell

Symbiotic microbes which are beneficial to many plants may become contaminants in the culture media (Herman, 1990). It has been reported that endogenous or endophytic fungi become pathogenic to the host plants when the plants are stressed, for example, when the cell walls are weakened or under other unfavorable *in vitro* conditions (Darworth & Callan, 1996). The endogenous or endophytic microbes are often hard to decontaminate. According to Cassells (1991), culture asepsis is important in all plant culture protocols. However, many trees in the tropics live and survive in association with endogenous or cryptic microbes (fungi) in the wild (Darworth & Callan, 1996). It remains unclear whether this association may continue to be mutual or lethal to the growth of plant cultures. This is because some fungi may not be '*vitropathogenic*', despite being prolific in the cultures (Herman, 1990).

association also breaks once the host plant is stressed.

**2.1.1 Exogenous fungal contaminants** 

injury. This can easily cause death of explants.

**2.1.2 Endogenous fungal contaminants** 

plant culture protocols.

propagation and mass multiplication of useful plants have failed. To a great extent, asepsis has always been a key factor towards successful *in vitro* plant culturing and mass multiplication. However, there have been limited research studies undertaken to document the success or failure of asepsis in many *in vitro* cultures of tropical plants.

There are several types of *in vitro* culture contaminants. They include various types of fungi, bacteria, viruses and other micro-organisms. Among these *in vitro* contaminants, fungi have been the most common and conspicuous microbes that cause many *in vitro* plant culture contaminations. In this chapter, we mainly outline a few common fungicides and other antibiotics (such as sterilants and/or disinfectants) and procedures employed to eliminate or reduce fungal *in vitro* culture contaminations to a manageable level as proliferation of fungi can cripple the whole plant culture process. We further highlight some common surface sterilants and disinfectants frequently utilized in plant culture laboratories to reduce or destroy fungal contaminants. In this respect, disinfectants and surface sterilants are chemicals used in plant culture laboratories to control or kill pathogens which may not necessarily be fungi. These surface sterilants or disinfectants may not be purely fungicides although they act against fungi. This chapter also presents the efficacy, handling, storage and the possible effects of fungicides on plant cultures and the roles of these fungicides in stock plant preconditioning to improve the efficacy of fungicides or other disinfectants. However, details of other forms of *in vitro* contaminants (such as bacteria, viruses and other micro-organisms) are outside the scope of this chapter.

#### **2.** *In vitro* **culture pathogens**

There are several pathogens (microbial contaminants) which have been a major threat to *in vitro* cultures due to their rapid proliferation characteristics (Enjalric et al., 1998). Axenic cultures are often mandatory throughout the entire *in vitro* culture process. The contaminated cultures are usually discarded at any stage of sub-culturing because many *in vitro* culture contaminants eventually cause plant death by exuding their phytotoxins (George, 1993).

Many fungal contaminants are visible right from the primary *in vitro* plant culture initiation except for the cryptic contaminants. Generally, these contaminants overgrow plant cultures and often outcompete them for resources in the nutrient media (Pierik, 1987). Consequently, *in vitro* contaminants often adversely interfere with culture growth and survival. Therefore, freedom of pathogens (asepsis) in plant cultures has been the ultimate goal in many *in vitro* protocols.

#### **2.1 Fungal contaminants**

Fungal contaminants have posed a major threat at every stage of the *in vitro* plant culture process. These fungal contaminants are characterized by the presence of hyphae (threadlike) appearing on the explants and culture media. Colonization of fungi progresses with time and the plant cultures are eventually covered in fungal mycelia. Unlike other contaminants, fungal contaminants are visible in the culture media. Furthermore, the development and growth of fungal contaminants are faster than the growth of the plant cultures. However, the rapid proliferation of fungi in the plant cultures depends on the type of fungi colonizing the culture media.

The fungi colonizing the plants could be endogenous, cryptic or endophytic. Herman (1990) reported that endogenous or endophytic microbes are often difficult to decontaminate. It is known that many endophytes are beneficial for the growth of ortet plants in the wild, but they also become contaminants in culture media. Helander et al. (1996) reported that mutualism between endophytes and the host plant depends on the prevailing plant condition. Endophytes can be harmful to plant cultures although they are useful outside the plant culture laboratories. Examples of some trees that live and thrive with symbiotic microbes (mycorrhizae) include *Uapaca kirkiana* (Mwamba, 1995), but this mutualistic association also breaks once the host plant is stressed.

In the tropics, fungal contaminants are very common due to the prevailing favorable environmental conditions (especially the high temperatures and humidity) for the proliferation of fungal pathogens on the ortet plants. Furthermore, the conditions in the plant culture incubation chambers are favorable for the rapid growth of fungi and other *in vitro* contaminants. For instance, the range of temperatures (usually between 23 – 26 oC), high humidity and plant nutrients (available inside the culture vials or test-tubes) cannot deter or inhibit the proliferation of many fungi in the tubes placed in the culture incubation chambers. Moreover, some plants live and survive by mutual association with fungi in the wild. However, such fungi are likely to become '*vitropathogenic*' in the plant cultures.

### **2.1.1 Exogenous fungal contaminants**

246 Fungicides for Plant and Animal Diseases

propagation and mass multiplication of useful plants have failed. To a great extent, asepsis has always been a key factor towards successful *in vitro* plant culturing and mass multiplication. However, there have been limited research studies undertaken to document

There are several types of *in vitro* culture contaminants. They include various types of fungi, bacteria, viruses and other micro-organisms. Among these *in vitro* contaminants, fungi have been the most common and conspicuous microbes that cause many *in vitro* plant culture contaminations. In this chapter, we mainly outline a few common fungicides and other antibiotics (such as sterilants and/or disinfectants) and procedures employed to eliminate or reduce fungal *in vitro* culture contaminations to a manageable level as proliferation of fungi can cripple the whole plant culture process. We further highlight some common surface sterilants and disinfectants frequently utilized in plant culture laboratories to reduce or destroy fungal contaminants. In this respect, disinfectants and surface sterilants are chemicals used in plant culture laboratories to control or kill pathogens which may not necessarily be fungi. These surface sterilants or disinfectants may not be purely fungicides although they act against fungi. This chapter also presents the efficacy, handling, storage and the possible effects of fungicides on plant cultures and the roles of these fungicides in stock plant preconditioning to improve the efficacy of fungicides or other disinfectants. However, details of other forms of *in vitro* contaminants (such as bacteria, viruses and other

There are several pathogens (microbial contaminants) which have been a major threat to *in vitro* cultures due to their rapid proliferation characteristics (Enjalric et al., 1998). Axenic cultures are often mandatory throughout the entire *in vitro* culture process. The contaminated cultures are usually discarded at any stage of sub-culturing because many *in vitro* culture contaminants eventually cause plant death by exuding their phytotoxins

Many fungal contaminants are visible right from the primary *in vitro* plant culture initiation except for the cryptic contaminants. Generally, these contaminants overgrow plant cultures and often outcompete them for resources in the nutrient media (Pierik, 1987). Consequently, *in vitro* contaminants often adversely interfere with culture growth and survival. Therefore, freedom of pathogens (asepsis) in plant cultures has been the ultimate goal in many *in vitro*

Fungal contaminants have posed a major threat at every stage of the *in vitro* plant culture process. These fungal contaminants are characterized by the presence of hyphae (threadlike) appearing on the explants and culture media. Colonization of fungi progresses with time and the plant cultures are eventually covered in fungal mycelia. Unlike other contaminants, fungal contaminants are visible in the culture media. Furthermore, the development and growth of fungal contaminants are faster than the growth of the plant cultures. However, the rapid proliferation of fungi in the plant cultures depends on the type

the success or failure of asepsis in many *in vitro* cultures of tropical plants.

micro-organisms) are outside the scope of this chapter.

**2.** *In vitro* **culture pathogens** 

(George, 1993).

protocols.

**2.1 Fungal contaminants** 

of fungi colonizing the culture media.

Generally, exogenous fungal pathogens are easy to eliminate from the stock plants using many fungicides. This is because such pathogens are found on the outer surface of the stock plants, and hence adequate contact between the fungicides and the pathogens and even the spores. However, insufficient surface disinfection might not dislodge exogenous pathogens and spores from the stock plants, and hence endogenous contaminants still pose a threat in plant culture protocols.

It is always difficult to detect the presence of either exogenous, endogenous or both pathogens on the stock plants. So it might be advisable to use systemic fungicides (e.g. Benomyl) before other disinfectants such as sodium hypochlorite are used. It is also a good practice to ensure that all possible culture contaminants are dealt with at the initial decontamination stage instead of re-disinfecting the explants at later stages (sub-culturing stages). Re-decontamination of explants might weaken the ensuing explants due to cell injury. This can easily cause death of explants.

#### **2.1.2 Endogenous fungal contaminants**

Symbiotic microbes which are beneficial to many plants may become contaminants in the culture media (Herman, 1990). It has been reported that endogenous or endophytic fungi become pathogenic to the host plants when the plants are stressed, for example, when the cell walls are weakened or under other unfavorable *in vitro* conditions (Darworth & Callan, 1996). The endogenous or endophytic microbes are often hard to decontaminate. According to Cassells (1991), culture asepsis is important in all plant culture protocols. However, many trees in the tropics live and survive in association with endogenous or cryptic microbes (fungi) in the wild (Darworth & Callan, 1996). It remains unclear whether this association may continue to be mutual or lethal to the growth of plant cultures. This is because some fungi may not be '*vitropathogenic*', despite being prolific in the cultures (Herman, 1990).

Efficacy and Utilization of Fungicides and Other Antibiotics for Aseptic Plant Cultures 249

Stock plants, especially from the wild are often difficult to decontaminate. For instance, *Uapaca kirkiana*, one of the wild trees of southern Africa, has been a difficult plant to eliminate *in vitro* fungal contaminants, especially when derived from mature ortet plants (Mng'omba et al., 2007). Success in culture asepsis of *U. kirkiana* has been achieved using seedling stock plants and 3.5% sodium hypochlorite (NaOCl) as a surface disinfectant (Maliro, 1997). However, they did not achieve any success in decontaminating adult *U.* 

Plants that live in association with endogenous fungi require preconditioning before collection. Application of Benomyl (Benlate), a systemic fungicide has been found effective on ortet plants before stock plant collection for *in vitro* culture. For instance, this method has been an effective way in reducing fungal load on *U. kirkiana* (Mng'omba et al., 2007). However, this process was time consuming as it involved isolation of grafted plants and placing in a screen or greenhouse where Benlate solution was regularly applied to eliminate fungi and the spores. The stock plants collected from such isolated plants are often free from heavy fungal infestation. It is advisable to avoid watering the whole ortet plants, but watering should be done to the polyethylene bags through drip irrigation or a hose pipe as

To increase the chances of eradicating fungal pathogens and spores on the mother plants, pruning might be necessary to induce proliferation of new and rejuvenated lateral shoots which may not be highly loaded with fungal pathogens, and hence ease decontamination unlike the old plant shoots. Furthermore, the prevailing weather conditions during collection of stock plants play a vital role. Collecting stock plants from the forests or open field on a rainy day increases fungal load on the stock plants, and hence makes it difficult to eliminate the pathogens as this would require a rigorous decontamination process. It is, therefore, clear that fungicides and other disinfectants do play a vital role in the entire *in* 

Surface sterilants can be described as chemicals rendering plants free from any pathogens including fungal spores. Generally, surface sterilants act on the outside of the explants. There are several chemicals which have been used to free plants from pathogens before culture initiation onto the media (chemotherapy). They deter growth and proliferation of pathogens (fungi, bacteria and other types of microbes). In many cases, a combination of several surface sterilants, disinfectants and/or fungicides is used to improve the efficiency of subsequent fungicides in decontaminating stock plants. For instance, a few drops of Teepol (0.05%) are often applied to the water and this increases wettability of the plant surfaces. Also, penetration of fungicides into the outer plant cells might be enhanced by rinsing the stock plants in the water. Generally, Teepol (soapy water or detergent) has been one of those disinfectants or surface sterilants commonly used in many plant culture laboratories to enhance the removal of pathogens and/or fungal spores from the stock

Another common disinfectant in plant culture laboratories has been ethanol (an alcohol derivative). Ethanol has been one of the commonest disinfectants used in plant tissue culture to eliminate pathogens and spores. It is used from the preparation room up to the laminar

*kirkiana* plant materials due to high fungal contamination at the initial stage.

most fungal pathogens and spores proliferate on wet plant surfaces.

*vitro* plant culture.

plants.

**4. Surface sterilants and disinfectants** 

Fungal contaminants increase plant culture mortality as they compete for the same resources (nutrients and oxygen) in the culture media or produce phytotoxins to harm the plant cultures. Obuekwe & Osagie (1989) reported that fungi such as *Aspergillus niger* and *Aspergillus flavus* produce oxalate and aflatoxin poisons respectively that can cause death to plant cultures. To overcome the fungal contamination problem, commercial fungicides have been used to control *in vitro* fungal contaminations (Table 1).


Table 1. Common fungicides and disinfectants used in plant culture laboratories

Establishing complete axenic cultures has not always been easy since some potential culture contaminants, especially endophytes mutually co-exist with plants. It is accepted that the success of achieving culture asepsis depends on the nature of stock plants used. This is because mature stock plants are highly loaded with fungal pathogens and spores, but mature stock plants are selected due to their fruiting precocity, especially for fruit trees. To achieve plant culture asepsis for such mature stock plants, there is need for pre-conditioning of ortet plants before collection of stock plants.

#### **2.1.3 Antibiotics**

Although the main focus of this chapter is on fungicides, antibiotics also play a vital role in achieving aseptic *in vitro* cultures. It has been observed that the use of fungicides alone can lead to proliferation of other *in vitro* contaminants such as yeast, bacteria, protozoa and other microbes (www.phytotechlab.com) hence broad-spectrum antibiotics are used in plant culture to control many *in vitro* contaminants. Other disinfectants and surface sterilants may not be necessarily fungicides but broad-spectrum antibiotics which are often used.
