1 Days after sampling refer to the day when aphids were in contact with the soil.
- = Soil not tested.

4.7 Conclusions

The population interaction between aphids and fungi from Entomophthorales is complex. The fungi need winter survival structures and the fungi must furthermore adapt to the aphid population biology, which may include alternating summer and winter hosts. The initiation of infection during spring, however, is very important since it may be the determinant for the spring and early summer prevalence levels in cereal aphid populations.

In summary, we may conclude:

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

Isolation

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.

Growth

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

5.3.1 Isolation

Isolation

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.

Carbon

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.

Nitrogen

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).

Vitamins and salts

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).

Serum

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

Insect haemolymph

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.

Temperature

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.

pH

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

Background

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.

Objectives

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.

Methods

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.

Results

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:

6. Virulence of Pandora neoaphidis against Sitobion avenae and Rhopalosiphum padi

6.1 Bioassay methodology

Pathogenicity and virulence

Pathogenicity of an insect pathogen is defined as the ability to produce disease in insects (Lacey, 1997). The proof of pathogenicity is the first step towards studies on virulence. The virulence of an insect pathogen is defined as the quality or property of being virulent (Lacey, 1997). Assessment of the virulence of an insect pathogen requires quantitative studies on dose-response relationships.

LC₅₀ and LD₅₀

In studies of dose-response relationships, the terms LC₅₀ and LD₅₀ are the most common expressions of virulence. LC₅₀ is the concentration of a given insect pathogen required to kill 50 % of the test insect population within a given period of time, whereas LD₅₀ expresses the dose required to kill 50% of the population. With respect to fungi from Entomophthorales, LC₅₀ is the appropriate term since the methodology only allows an estimate of the concentration used and not the dose actually received by the test insects (Papierok & Hajek, 1997).

LT₅₀

The term LT₅₀ is defined as the time period required to kill 50 % of the test insect population when subjected to a given concentration or dose of an insect pathogen. The term is often used a quantitative expression of the virulence of fungi from Entomophthorales. The shorter the lethal time documented for a fungus against the test insect, the higher the virulence. Furthermore, the term LT₅₀ provides much of the information needed to understand the dispersal of the disease in the insect population and the dynamics of the host-pathogen system. In several cases, both LC₅₀ and LT₅₀ were measured for a host-pathogen system using a fungus from Entomophthorales as the pathogen and aphids as the test insect (Papierok & Hajek, 1997).

Bioassay methodology

Bioassays with Entomophthorales can be performed in different ways. The most common method is the ‘conidia shower’ method (Papierok & Hajek, 1997, Eilenberg, 1999). The test insects are subjected to a conidial shower (sporulating cadavers or conidia from in vitro cultures). The concentration of conidia is expressed as number of conidia per mm² or similar. Using insect cadavers, less precise expressions such as number of cadavers are sometimes used to express the concentration of conidia. Conidial showers from cadavers or in vitro cultures do in any case reflect the natural route of infection, although studies using Entomophthorales may suffer from limited replicability.

Injections into the insect haemolymph of protoplasts or hyphal bodies have also been used to assess virulence (Papierok & Hajek, 1997). This method gives high replicability but does not reflect the natural route of infection.

6.2 Bioassays against Sitobion avenae and Rhopalosiphum padi

LC₅₀ of P. neoaphidis against S. avenae

Our experimental determination of the LC₅₀ of P. neoaphidis against S. avenae was carried out using an in vitro isolate of P. neoaphidis and the conidia shower method. Cohorts of ten aphids from two clones (green or brown alates or apterous) were subjected to a conidial shower. We varied the time under conidial shower (0, 5, 10, 15, 25, 35, 50, 80, 120, 150 and 180 minutes) to obtain different concentrations of conidia, and the number of conidia per mm² was calculated. Incubation took place at 18^oC and mortality was recorded after seven days.

Data are shown in table 6.1. There was no significant difference between the two clones. For both clones the LC₅₀ values for alates were significantly lower than for apterae.

Table 6.1
Susceptibility of morphs and clones of Sitobion avenae to infection of Pandora neoaphidis (Isolate KVL-634), expressed by LC₅₀ values

LT₅₀ of P. neoaphidis against S. avenae

The experiments to assess LT₅₀ were performed using a similar methods as in the LC₅₀ studies. All test insects were however subjected to conidia for 60 minutes before being placed in different temperatures. Data from the experiments using green alates are shown in figure 6.1.

Figure 6.1 Look here!
Lethal time (LT₅₀) of alates of the green Sitobion avenae clone (H1) infected with in vivo material of Pandora neoaphidis (Isolate KVL-634).

As seen in figure 6.1, lethal time is highly dependent on temperature. At 25^oC, the lethal time is approximately 4 days, while incubation at 5^oC results in a lethal time of 12-14 days.

A comparison of the lethal time at 18^oC for S. avenae subjected to P. neoaphidis is seen in table 6.2. The calculated lethal times varied between 6.6 and 7.5 days between the morphs and clones, though the differences were not significant.

Table 6.2
Lethal time at 18^oC of morphs and clones of Sitobion avenae infected with in vivo material of Pandora neoaphidis (Isolate KVL-634).

Virulence of P. neoaphidis against R. padi

Similar experiments were carried out using R. padi as the receptor. These data are included in table 6.3.

Table 6.3
LT₅₀ data with aphids and Entomophthorales from our experiments and from the literature.

LT₅₀ data on aphids/ Entomophthorales

Table 6.3 shows our data on LT₅₀ along with the literature data for P. neoaphidis or other fungi from Entomophthorales and different aphid species. The three fungus species are the most common species on cereal aphids. As can be seen in the table, the generel tendency is that at incubation temperatures above 20^oC the lethal time is 3-5 days, whereas it is much higher at lower temperatures. Our data on S. avenae give precise information on this relationship and also contributes to knowledge concerning the general understanding of aphid/fungus relationships.

6.3 Conclusions

Discussíon and conclusions

The bioassay methodologies used for Entomophthorales were modified in order to obtain precise data on S. avenae and R. padi. The method allowed a comparison between different morphs and clones for the aphids, and the data obtained were satisfactory with respect to reproducibility.

A comparison of our data with the literature data (table 6.3) demonstrated that aphids are quickly killed by fungi from Entomophthorales, but there are differences in the measured LT₅₀ data depending on aphid species, aphid morph, fungus species or even isolate and incubation temperature.

In summary, we may conclude:

7. Dynamics of Pandora neoaphidis epizootics in Sitobion avenae populations

7.1 Definitions of some model terms

Models

An understanding of the dynamics of fungal diseases of insects is critical to the development of epizootiological theories as well as potential prediction of infection levels for pest management purposes (Hajek et al., 1993). A typical integrated pest management (IPM) system model consist of three parts: (1) a biological-conceptual model developed from the literature, (2) a mathematical representation of that framework, and (3) a computer program implementing the mathematics (Brown, 1987).

However, the dynamics of fungal epizootics are poorly understood, and factors necessary for the development of epizootics have only been identified for very few host-pathogen systems (Hajek, & St. Leger, 1994). As far as we know, only one IPM model takes entomopathogenic fungi into account, namely a system consisting of the cotton aphid (A. gossypii) and N. fresenii in Arkansas, USA (Steinkraus, 1998) which is a system quite different from the cereal system in Denmark.

7.2 A biological conceptual model for the dynamics of Pandora neoaphidis in Sitobion avenae populations

A biological conceptual model

The dynamics of P. neoaphidis in populations of S. avenae are illustrated using a flow diagram in figure 7.1. In the figure both the aphids and the fungus are divided into three ‘stage variables’ each according to the principle phases of the disease. These stage variables are connected with several ‘development rates’. The development rates are dependent on abiotic factors such as temperature, moisture and solar radiation (‘forcing variables’). Moreover the forcing variables may determine whether an infection can occur (Benz, 1987; Carruthers & Hural, 1990). Since most of the processes are occurring in a cereal field, the crop development as well as agricultural practices must be included as forcing variables in the model. Some pesticides have shown to have a negative impact on the development of epizootics (Zimmerman, 1978; Wilding & Brobyn, 1980). As discussed in chapter 4, soil is important as a reservoir of P. neoaphidis and thus for early development of epizootics with P. neoaphidis.

Figure 7.1 Look here!
Flow diagram of an epizootiological model of the dynamics of Pandora neoaphidis in a population of Sitobion avenae. Grey rectangles are stage variables, ovals are developmental rates and the two large rectangles are forcing variables.

7.2.1 Stage variable

Aphids

Aphids are divided into the following categories: susceptible healthy aphids (S), infected aphids (D) and sporulating cadavers which spread the infective conidia (I). The sporulating cadavers only exist long enough to release the infectious units and will then disperse from the system. In other models the insect population has been divided into as many as five categories according to the development of the disease within the insect (eg. Schmitz et al., 1993; Ardisson et al., 1997). However, the categories described here are those which can usually be estimated from field data.

In this model it is assumed that all host stages and morphs are equally susceptible to aphid pathogens. This is however a simplification since results analysed in this project demonstrated a difference in prevalence between alate and apterous S. avenae within cereal fields (Appendix D). Bioassays were conducted and it was shown that this difference in prevalence was due to differences in susceptibility rather than differences in behaviour. To keep the model as simple as possible, however this is not taken into account and may not be important for the dynamics since most alate aphid probably emigrate before they die from the disease.

Fungus

The pathogen is divided into survival structures (O), primary conidia (P) and secondary conidia (Q). The cadavers produce either conidia, or survival structures or both. The conidia are disharged actively from cadavers and cause secondary infections among the aphids. Whether the primary and / or the secondary conidium is the infective unit is still not known. However, for

E. muscae it has been proven that secondary conidia are about 200 times as infective as primary conidia (Bellini et al., 1992) and the same can be true in our system. As mentioned in chapter 4 the question of how P. neoaphidis survive is poorly understood. The fungus must however, overwinter in some stage and initiate primary infections in spring.

7.2.2 Effect of abiotic factors on development rates

Abiotic factors do not act independently but as a complex of processes. Therefore, analysis of single environmental factors on the epizootics of fungal diseases is not always successful. Moreover, true quantitative values are difficult to obtain in nature since the microenvironment of an insect and/or a pathogen may differ considerably from the average measurable conditions of the environment (Benz, 1987). The effects of abiotic factors on the dynamics of disease development are discussed in greater detail below.

Birth rate (a)

In spring S. avenae migrate to cereal fields when accumulated day degrees reach 1150-1250D^o (above a threshold of 0^oC) and they begin to reproduce (Hansen, 1995). The reproduction rate is primarily temperature dependent. Because only horisontal transmission of fungus diseases occurs, both susceptible and infected hosts produce susceptible nymphs which moult through a normal sequence of nymph instars and finally become adults. The fecundity decreases for an infected aphid compared to a healthy one. The closer the aphid gets to death the larger the decline in fecundity (Schmitz et al., 1993).

Migration rate (b)

It is assumed that the migration rate for infected aphids follows the migration rate for healthy aphids since information in the litterature concerning change in migration behaviour is found.

Infection rate (c)

Before infection of an aphid can occur, contact is necessary between the infective unit and the aphid. Tanada & Kaya (1993) reported that the spread of disease depends on both densities of host and infective unit. An increase in one or both pools enhances the probability of contact between spores and aphids. In contrast, Wilding (1975) found that aphid density had only minor importance in the transmission of infection. Missionnier et al. (1970) similarly concluded that epizootics were independent of host density. An analysis of Danish data has shown that density of aphids only has an impact when the density is low and that a threshold value probably exists. In addition to contact between spores and aphids, suitable temperatures and relative humidities are necessary for germination of spores and for penetration of the aphid integument (Benz, 1987).

Conidia of P. neoaphidis require a humid environment to be able to germinate and penetrate the integument. At high temperatures, a shorter time with free water is required to ensure optimal germination and penetration (Milner & Bourne, 1983). Germination occurs at any temperature between 10 ^oC and 20^oC (Milner & Bourne, 1983), and outside of this interval no results have been found in the literature.

Lethal time (e)

Our experiment showed that the lethal time increased as temperature decreased for aphids infected with P. neoaphidis. The relationship between lethal time and temperature can be expressed by the equation:

LT₅₀ = -6.05*ln (temp.) + 23.0

R² = 0.96

Production rate 1 (f)

Dromph et al. (1998) demonstrated a positive correlation between dry weight of S. avenae cadavers and total production of P. neoaphidis primary conidia at 18^oC. The relationship can be expressed as:

Conidia produced = 3186*dry weight (in mg) + 11412

R² = 0.97

Climatic factors, particularly humidity and temperature act on conidia discharge (Benz, 1987). Dromph et al. (1998) demonstrated that temperature had a significant impact on sporulation, particularly on total production, which increased with temperature. At 18^oC most conidia were produced within the first twelve hours following death of the aphid. Wilding (1969) proved that

P. neoaphidis only sporulated when RH was at least 90 %. Within this range, the numbers of spores increased with increasing humidity. Light apparently does not affect the sporulation of P. neoaphidis (Milner, 1981).

Production rate 2 (g)

It is assumed that climatic factors, particularly humidity and temperature act on the capacity of secondary conidia production in the same way as in primary conidia.

Production rate 3 (h)

No quantitative data have been collected concerning the initiation of the survival structure. Temperature and nutritional conditions may play an important role (Latgé & Papierok, 1988) and probably the day length too.

Production rate 4 (i)

For some entomophthoralean fungi a dormancy period is required prior to germination (Latgé & Papierok, 1988). Our data suggest that quiescence or dormancy for the survival structure of P. neoaphidis is broken a long time before the aphids arrive to the field. Infection can thus start as soon as the aphids arrive.

P. neoaphidis loses the infectivity at a rate dependent on humidity and temperature (Wilding, 1973; Brobyn et al., 1987). Inoculum on leaves near the base of the plants remained infective longer than on leaves near the top (Broby et al., 1985) probably as a result of less solar radiation.

7.3 Conclusions

In summary, we may conclude

8. General discussion and conclusions

8.1 Background

The background for this work was the need to develop alternative methods for pest insect control. Due to a general wish from the society to minimise the use of chemical pesticides, biological control may offer a sound, environmentally friendly alternative.

Choice of system

The cereal aphid/fungus system was chosen as a model system based on:

1) The importance of cereals in Denmark, 2) The difficulties in controlling aphids in cereals without chemical pesticides, and 3) Knowledge of the natural occurrence of entomopathogenic fungi obtained in an earlier project.

Aphids will in the future still be significant pest insects, and the desire to minimise the use of chemical pesticides has certainly not diminished during the project period (eg. the "Bichel Report" to the Ministry of Environment 1999).

The project proceeded in three ways: 1) New scientific information was obtained during the project period based on the specific experimental work, 2) Relevant literature was reviewed, and 3) A high level of information exchange was maintained with other research groups (in Denmark and internationally) during the period. The report contains both specific new data from our studies, a compilation of data from the literature, and the newest available data from other research groups, including the most recent findings reported at conference in August 1999.

8.2 General Perspectives

The national dimension

For the Danish cereal system, the set of data obtained represents a solid basis for future work directly addressing biological control. Two types of biological control are potentially possible: 1) A direct release of Entomophthorales as biopesticides and 2) An enhancement of the natural control of aphids with Entomophthorales. Both strategies were given attention in our studies. With respect to 1), the fungus P. neoaphidis has the highest potential at present. We were able to characterise, isolate and grow this fungus in vivo and in vitro, perform bioassays and develop parts of a model for this species in relation to cereal aphids.

However, before the fungus can be used directly as a biopesticide, a range of further experiments must involve: more efficient growth in vitro, formulation, release in the field, and evaluation of field release. Our studies have shown that the fungus should be subjected to further investigation in order to develop outdoor biological control.

Our attention on the winter survival of Entomophthorales provided us with significant information for a deeper understanding of the plant-aphid-fungus system. Based on the results we do not hesitate to propose that future use of Entomophthorales to control aphids should not solely be based on the biopesticide approach, but that strategy 2) should certainly also be seriously considered to enhance natural control (eg. earlier initiation of epizootics). Additional experimental work should in such cases include more in depth studies on the importance of cropping systems, surrounding hedges and other landscape structures for the development of epizootics.

The European dimension

During the project period, attention on Entomophthorales as biocontrol agents of aphids has increased significantly. In particular, the group at ETH, Switzerland, and the group at Rothamsted Exp. Station, UK, should be mentioned. We maintained close contact with these groups and performed experimental work in co-operation (e.g. growth in vitro). The increased attention on Entomophthorales has led to a European network cooperation from year 2000 (COST Action 842). Results from the Danish studies on cereal aphids contributed to the success of the application.

In the coming years, we plan to use the European network to ensure that our results will be used by the scientific society to assist in well-designed experiments for both the cereal aphid-Entomopthhorales system and for other insect-fungus systems as well.

In summary, conclusions from the project as a whole are as follows

9. References

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Appendix A

Morphological features of entomophthoralean fungi infecting aphid

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Appendix B

Random Amplified Polymorphic DNA- PCR

Isolation of DNA

Before isolation of DNA all fungi were grown for one to three weeks in Grace’s insect culture media added 5% Fetal Bovine Serum (Gibco BRL) at 20^oC in constant dark. The mycelium was harvest after centrifugation under sterile conditions and DNA was isolated following the protocol of Hodge et al. (1995) but modified by a RNase treatment. After precipitation of nucleic acid with isopropanol RNA was degraded by resuspending the nucleic acid in 250 m l sterile TE buffer (10 mM Tris-HCL; 0.1 nM EDTA). 1:125 RNase PLUS was added (5 Prime ® 3 prime, Inc.) and samples were incubated for 2 hours at 37^oC. Samples were then centrifuged at 13000 rpm at 5^oC for 20 minutes and ammonium acetate was added to a concentration of 5.0 M. DNA was precipitated overnight at –20^oC with 2.5 vol cold 95% ethanol, centrifuged at 13000 rpm and washed twice with 70% cold ethanol. DNA was resuspended in filter sterilised distillated water and quantified by UV spectrophotometry.

RAPD

Amplification reactions were performed in 1.5 ml centrifuge tubes with a total volume of 30 ? l. Each reaction contained 1x supplied PCR Buffer (10mM Tris-HCl, 10mM KCl, Gibco BRL), 3 mM MgCl₂, 200 ? M each of dATP, dCTP,dGTP and dTTP (Boeringer Mannheim), 0.5 ? M primer, 5 ng genomic DNA and 3 units Taq polymerase (Gibco BRL). Reactions were overlaid with 30? l mineral oil and amplifications were performed in a MJ Research PTC-100 thermocycler with the following cycle parameters: an initial denaturisation of 2 min at 93^oC; 40 cycles of 1 min at 93^oC, 1 min at 36^oC; 2 min at 72^oC; a final extension of 7 min at 72^oC. Amplification products were separated by electrophoresis in 1.4% agarose gels with 1x TBE buffer at 44 v for 18 hours and detected by staining with ethidium bromide. For each tested primer two replicates were carried out.

Lengths of DNA fragments were measured and scored from photographs of the gels. Differences in intensity were not taken into account and products not appearing in both replicates were disregarded. Data was recorded as a binary matrix and phenetic similarity was calculated (UPGMA using Jaccard’s coefficient) by use of the statistical software NTSYSpc (V.2.01e).

Appendix C

SOIL - A NATURAL SOURCE OF ENTOMOPHTHORALEAN FUNGI INFECTING APHIDS

Reprinted with permission from the International Organization for Biological and Integrated Control of Noxious Animals and Plants

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Appendix D

Poster presented at Society for Invertebrate Pathology 29^th Annual Meeting, September 5, 1996. Cordoba, Spain.

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