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Entomophthorales on cereal aphids

5. In vivo and in vitro isolation and growth of entomophthoralean fungi

5.1 Introduction

Information concerning factors important for in vivo and in vitro isolation and growth is important for both basic studies and the host-pathogen relationship as well as for the development of mass production methods.

5.2 In vivo isolation and growth

For some of the entomophthoralean fungi the only means for growing the fungus is by using living host insects. The first step in running such an in vivo culture of any Entomophthorales involves identification and isolation. Usually one cadaver is placed over a glass slide under humid conditions to allow sporulation. The fungus is then identified while it is ensured that only one fungus species has invaded the insect tissue. Thereafter the cadaver can be used to start an in vivo culture. Preferably a culture is always initiated from a single cadaver to obtain an isolate which is as homogeneous as possible.

Methods for in vivo culturing of Entomophthorales are described for example in Entomophthora schizophorae Keller & Wilding in Keller infecting flies (Kramer & Steinkraus, 1981; Eilenberg, 1987) and Neozygites floridana (Weiser & Muma) Remaudière & Keller infecting mites (Smitley et al., 1986). The principle is to permit conidia produced from sporulating cadavers to fall into a cage with healthy insects or onto plant material and then later add healthy insects. After some days, depending on temperature, host species and fungus, some of the insects exposed to condia die as a result of the fungus and begin to sporulate. A new cycle of disease can then be started from these insects.

5.2.1 In vivo culturing of Pandora neoaphidis infecting aphids

Pandora neoaphidis

The method for maintaining an in vivo culture of P. neoaphidis on S. avenae developed in this project is illustrated in figure 5.1.

Figure 5.1
Set-up for in vivo transmission of Pandora neoaphidis to healthy aphids.

Between one and four sporulating cadavers of S. avenae were fixed with vaseline to the lid of a 25-ml plastic cup with 3% water agar in the bottom to keep humidity high. Six to ten healthy aphids were then transferred to a straw of winter wheat placed in the water agar. The cup was closed and incubated at 17-20^oC. From day four following inoculation the aphids in the cup were checked daily and infected aphids were removed and used to establish a new cycle of disease. One of the most important matters in this system is to maintain humidity close to 100% (Wilding, 1969).

5.3 In vitro isolation and growth

The first attempts to cultivate entomophthoralean fungi in vitro were done by discharging conidia from a cadaver into different kinds of media (Sawyer, 1929; Müller-Kögler, 1959; Gustafsson, 1965). Rockwood (1950) was the first to isolate P. neoaphidis by transferring sporulating hyphae to an egg medium as early as 1934. MacLeod (1956) developed a new method for isolating Entomophthorales where he surface sterilised non-sporulating cadavers and transferred the whole insect to the medium (figure 5.2). For small insects such as aphids this method is still recommended (Keller, 1994; Papierok & Hajek, 1997).

Figure 5.2
In vitro isolation of Entomophthorales using the whole cadaver method (modified after Keller, 1994).

Isolation from surface sterilised resting spores formed inside a cadaver has also been reported (Tyrell & MacLeod, 1975; Papierok & Hajek, 1997). Finally in vitro isolation has been carried out from the vegetative stages such as protoplasts or hyphal bodies by collecting small amounts of haemocoel with a syringe from an infected but still living insect (Papierok & Hajek, 1997).

For isolation both liquid and solid media have been used. Some species, eg. E. muscae, are best isolated in liquid media (Eilenberg et al., 1992).

5.3.2 Nutritional requirements for in vitro growth

First attempts to grow Entomophthorales in vitro

Classical media, such as Sabouraud dextrose agar (SDA) or Sabouraud maltose agar (SMA), are not suitable for the majority of Entomophthorales. Sawyer (1929) and later Müller-Kögler (1959) both recommend coagulated egg yolk from hens as the best substrate for in vitro cultivation. Müller-Kögler (1959) also succeeded to in growing an unidentified species of Entomophthoraceae on milk agar, oat milk agar, beef extract, peptone yolk agar and potato pieces, however growth was never as significant as on the coagulated egg yolk media. Media based on coagulated egg yolk are still very common for isolation and growth of many species of Entomophthorales. Studies on the influence of the different lipid and protein fractions of egg yolk on the growth of several species of Entomophthorales proved that egg yolk does not contain any specific nutrients qualitatively required for Entomophthorales (Latgé & Bièvre, 1976; Latgé et al. 1978). Later Latgé (1982) concluded that Entomophthorals primarily grow well on egg yolk because it is highly concentrated in nutrients with a water content of only 50% and furthermore because highly concentrated carbon is not linked to high osmotic pressure.

Glucose has primarily been used as the carbon source, although maltose, fructose, thalose and glycerol have also been shown to be acceptable whereas sucrose is not (Latgé, 1975; Latgé et al., 1978). In some cases sources other than glucose have been shown to be superior. This is for example the case for Batkoa apiculata (Thaxter) Humber and Erynia curvispora (Nowakowski) Remaudière & Hennebert (Gustafsson, 1965).

Other carbon sources may be used in some cases. Latgé and Bièvre (1976) reported that C. obscurus, C. thromboides and others were able to use fatty acid as the carbon the source.

Optimum growth is obtained on complex media of amino acids or protein hydrolisates as nitrogen sources. (Latgé, 1975). No species are able to utilise nitrate as the nitrogen source (Gustafsson, 1965; Latgé, 1982).

Latgé & Sanglier (1985) showed for C. obscurus that Mg and to lesser extent Zn and Mn stimulated the formation of azygospores. They also showed that sulphur must be added in a reduced or oxidized form and phosphate must be present in the culture medium. Vitamins do not seem to have a significantl impact on growth (Dunphy & Nolan, 1982).

For some Entomophthorales, fetal serum has been shown to be essential, especially for those fungi which are growing as protoplasts. (Dunphy & Nolan, 1979; 1982).

Grundschober et al. (1998) showed that insect haemolymph was mandatory for sustained growth of Neozygites parvispora (MacLeod & Carl) Remaudière & Keller, a pathogen to many thrips. This means that for some entomophthoralean fungi at least one putative growth factor is present in the haemolymph. The growth factor however has not yet been found.

The nutritional requirements for in vitro growth of insect pathogenic Entomophthorales are summarised in figure 5.3.

Figure 5.3
General table of nutritional requirements for in vitro growth of Entomophthorales infecting insects (modified after Latgé, 1982). Erynia sensu lato includes species belonging to the genera Erynia, Pandora and Furia.

5.3.3 Physical requirements for in vitro growth

Solid contra liquid media

Routine in vitro culturing depends on the developmental stage of the fungus required. Hyphal bodies grow both on solid and in liquid media while protoplasts only grow in liquid media (Papierok & Hajek, 1997). Entomophthoralean species growing only in vitro as protoplasts are necessarily kept in liquid media. Other species, which represent the majority, can be maintained on solid media.

Gustafsson (1965) determined the temperature cardinal points for Pandora dipterigena (Thaxter) Humber, P. nouryi, P. neoaphidis and Conidiobolus thromboides on solid media and found that growth for all species was better at 21^oC and 24^oC than at 5 ^oC, 10 ^oC and 28^oC. At 28^oC the growth of all strains of P. neoaphidis was either very poor or absent. It is however remarkable that P. neoahidis grew even at 5^oC. Robinson (1986) determined that the optimal temperature for colony radial growth of P. neoaphidis was 20^oC.

In general Entomophthorales are tolerant of pH levels between 6 and 7 with an optimum of approximately 6.5 for the species investigated so far. At pH below 5 and above 8 growth is remarkably depressed (Gustafsson, 1965; Latgé et al. 1977; Dunphy & Nolan, 1979; Latgé & Sanglier, 1985; Robinson, 1986). For P. neoaphidis the fastest growth rate has been obtained between pH 6.0 and 7.0 (Gustafsson, 1965; Robinson, 1986). The optimal pH corresponds well with the pH usually found in insect haemolymph. Gustafsson (1965) concluded that P. neoaphidis has a relatively narrowly limited pH optimum compared with species from the genus Conidiobolus.

5.3.4 Vegetative growth of Pandora neoaphidis in liquid cultures

Earlier studies have shown that it is possible to cultivate P. neoaphidis in vitro on both solid media and submerged in liquid media for most of the media mentioned above. For commercial use of P. neoaphidis, liquid media are preferable due to faster and more homogeneous growth of the fungus compared to solid cultures. However, many problems are still to be resolved before the fungus can be used as a biopesticide. One of the most significant problems of growing P. neoaphidis in liquid media is the formation of pellets and / or heavy wall growth on the culture flasks (Robinson, 1986; Gray, 1990). The factors, which are supposed to influence the uneven growth are both mechanical and physiological. Gray (1990) suggested that an increasing oxygen concentration dissolved in the media decreases the degree of pellet formation. Formation of pellets can also be prevented by disruption of the inoculum in the flasks (Gray, 1990). This may be done either by using baffles in the culture flasks or by increasing the agitation speed. This may simultaneously increase the amount of dissolved oxygen in the media. Finally the source/morphology of the inoculum may play a significant role.

The primary objective of this investigation was to elucidate the effects of mechanical stress by using baffles and different agitation speeds (60, 120, 180, 240 rpm). Furthermore, the effects of inoculating liquid media with different sources of inoculum (conidia, hyphal fragments from a liquid culture or homogenised pellets from a solid culture) were also investigated.

The effects were measured as weight of biomass after different times of growth, glucose concentrations in the filtrate mentioned above, measurements of length of hyphal bodies, ability to sporulate and finally a visual determination of wall growth and pellet formation. One isolate of

P. neoaphidis (KVL 98-11) was chosen for these experiments. The isolate was isolated from R. padi in Copenhagen, Denmark, in the summer of 1998 and maintained in vitro since. In all experiments liquid cultures were grown in YEMG containing 1.6% (w/v) glucose (BioChemica), 1.0% (w/v) yeast extract (Oxoid) and 10% (v/v) pasteurised semi-skimmed milk (Coop). All cultures were grown at 20^oC in constant darkness.

To elucidate the effects of the source of inoculum liquid media was inoculated with conidia, hyphal fragments from a liquid culture or homogenised pellets from a solid culture. Best results with respect to fast and homogeneuos growth were obtained when the liquid culture was inoculated from another liquid culture. When inoculating with conidia, growth started very slowly and it took more than a week before growth was detectable. When inoculating with homogenised pellets from a solid culture, many fungal cells were destroyed indicating that much more inoculum must be used in order to obtain good and fast growth. Furthermore, growth was not more homogeneuos than when using liquid culture for inoculation.

The effect of baffles was a greater speed of growth at all four agitation speeds (Figure 5.4), however very heavy wall growth occurred particularly at the highest agitation speeds. After 180 hours of growth at 240 rpm the wall growth reached the lid of the flasks. This suggests that other types of baffles should also be tested.

For flasks without baffles speed of growth was very slow at 60 and 120 rpm, whereas the speed of growth was acceptable at both 180 and 240 rpm.

As shown in table 5.1 agitation speed also had a significant effect on sporulation when measured as number of conidia per mm² over 24 hours.

During the 144 hours that the experiment took place no remarkable changes in the distribution of length of hyphal fragments were observed (figure 5.5).

Table 5.1
Effects of agitation speed and baffles on sporulation of Pandora neoaphidis over 24 hours (isolate KVL 98-11) in YEMG in 20 ml flask culture (100 ml Erlenmeyer flask, 20^oC; constant darkness.). wb = with baffles; ob = without baffles.

Figure 5.4 Look here!
Effects of agitation speed and baffles on growth of Pandora neoaphidis (isolate KVL 98-11) in YEMG in 20 ml shake flask cultures (100 ml Erlenmeyer flasks; 20^oC; constant darkness).

Figure 5.5 Look here!
Effect of agitation speed and baffles on growth of Pandora neoaphidis (isolate KVL 98-11) in YEMG in 20 ml flask cultures (100 ml Erlenmeyer flasks; 20^oC; constant darkness).

5.4 Formulation and application

The use of P. neoaphidis for augmentative biological control by dispersing sporulating cadavers or infected aphids gave mixed results (Wilding, 1981; Wilding et al., 1990). Another and probably less laborious method is to use in vitro produced material. Latteur & Godefroid (1983) and later Sylvie et al. (1990) conducted a number of experiments where unformulated hyphal bodies were sprayed to control aphids. However, in none of the cases was adequate control obtained.

Very recently a Swiss group of insect pathologists began working on formulating Entomophthorales, primarily P. neoaphidis. They found that encapsulating hyphal bodies into sodium alginate beads gave promissing results concerning conidiation and infectivity against aphids in the laboratory (Shah et al. 1998). Glasshouse experiments carried out by the same Swiss group have shown promising results concerning biological control of the potato aphid (Macrosiphum euphorbiae (Thomas) with alginate formulated P. neoaphidis (Tuor et al., 1999).

5.5 Conclusions

In summary, we may conclude:

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