**3. Fungicides for stock plant pre-conditioning**

Fungicides are often used to reduce the pathogen loads from the stock plants to achieve culture asepsis in the culture laboratories. However, attention must be paid to the source of stock plants as many fungal contaminants are derived from the ortet plants. A few are then introduced during the culture initiation or at later stages of *in vitro* plant culturing. Leifert (1990) reported that sub-culturing processes are potential sources of contaminants and it is estimated that 5 - 15% of contaminants are introduced per sub-culture. This possibly suggests that a large number of fungal contaminants originate from the plants themselves. Other possible sources of fungal culture contamination have been due to poor handling of plant cultures during excision, initiation and sub-culturing and insufficient test tube sterilization and poor media preparation. These potential sources of fungal culture contaminations can be avoided with proper care when handling plant cultures.

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

sterilant

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

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

Fungicides are often used to reduce the pathogen loads from the stock plants to achieve culture asepsis in the culture laboratories. However, attention must be paid to the source of stock plants as many fungal contaminants are derived from the ortet plants. A few are then introduced during the culture initiation or at later stages of *in vitro* plant culturing. Leifert (1990) reported that sub-culturing processes are potential sources of contaminants and it is estimated that 5 - 15% of contaminants are introduced per sub-culture. This possibly suggests that a large number of fungal contaminants originate from the plants themselves. Other possible sources of fungal culture contamination have been due to poor handling of plant cultures during excision, initiation and sub-culturing and insufficient test tube sterilization and poor media preparation. These potential sources of fungal culture

not be necessarily fungicides but broad-spectrum antibiotics which are often used.

contaminations can be avoided with proper care when handling plant cultures.

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

increases culture mortality both a fungicide and sterilant antibiotic toxic to fungi and yeast

fungicides - act as multiplication inhibitors fungicide - wide microbial activity (kills spores) oxidizing agents – kills wide range of pathogens

been used to control *in vitro* fungal contaminations (Table 1).

of ortet plants before collection of stock plants.

**3. Fungicides for stock plant pre-conditioning** 

Benomyl and Captain Formaldehyde (aldehyde) Sodium & calcium hypochlorite

Teepol (detergent) Mercuric chloride Copper oxychloride

Nystatin

**2.1.3 Antibiotics** 

Common chemicals Type and some functions

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

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 most fungal pathogens and spores proliferate on wet plant surfaces.

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 vitro* plant culture.
