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Entomophthorales on cereal aphids

4. Survival of entomophthoralean fungi during winter

Cereal aphids are only present each year from May through October in Denmark. This means that Entomophthorales which attack cereal aphids must survive in the environment for at least six months out of every twelve.

4.1 Survival structures

The fungus may survive either as (1) resting spores (Burger & Swain, 1918; Batko, 1964ab; Remaudière & Hennebert, 1980; Remaudière & Keller, 1980; Keller, 1987,1991; Ba»azy, 1993), (2) hyphal bodies (Keller, 1997a; Feng et al., 1992), (3) conidia or ‘loriconidia’ (Weiser & Batko, 1966; Latteur & Randall, 1986) or (4) in anholocyclic aphid populations (Byford & Reeves, 1969; Wilding, 1973). The survival strategy depends on the fungus species (table 4.1).

Table 4.1
Suggested survival structures for aphid pathogenic Entomophthorales

4.2 Resting spores

Among the aphid pathogenic fungi resting spores have been described as occurring in vivo for all species except P. neoaphidis, P. kondoiensis, Z. phalloides and Neozygites lageniformis (Thaxter) Remaudière & Keller (Thaxter, 1888; MacLeod & Müller-Kögler, 1973; Remaudière & Keller, 1980; Keller, 1987; Keller, 1991; Ba»azy, 1993). Only limited information is available for the last species mentioned, and resting spores may occur.

4.2.1 Development of resting spores

A variety of factors are found to promote the development of resting spore, including abiotic parameters such as temperature (Shimazu, 1979; Milner & Lutton, 1983; Glare et al., 1989; Hajek & Shimazu, 1996, Thomsen 1999), light (Thomsen, 1999), humidity (Glare, et al., 1989) and biotic factors such as the fungal isolate (Glare et al., 1989; Hajek, & Shimazu 1996; Thomsen, 1999), fungal density (Glare et al., 1989; Hajek & Shimazu, 1989) host (Ben Ze’ev & Uziel, 1979), host age (Wilding & Lauckner, 1974; Shimazu, 1979; Steinkraus & Kramer, 1989; Hajek & Shimazu, 1989) and sex of host (Thomsen, 1999)

4.2.2 Resting spore germination

The timing of resting spore germination, which can take place over a longer period seems to be correlated with host, pathogen, temperature, humidity and light (table 4.2). Often a period of cold is required. Nonetheless the exact requirements for initiating germination have not been completely elucidated. Resting spores can remain infective for several years under field conditions.

Table 4.2
Entomophthoralean resting spores dormancy requirements (modified after Hajek, 1997).

4.3 Hyphal bodies

Under cool and dry conditions it is possible for P. neoaphidis to survive for several months as hyphal bodies in cadavers without affecting the virulence of the conidia produced when the cadavers are moved to warmer and more humid conditions (Wilding, 1973; Courtois & Latteur, 1984; Latteur et al., 1985). Humidity and temperature in Denmark will probably never be consistently low enough during autumn and winter to ensure survival as hyphal bodies. However in cadavers of the pea aphid A. pisum, Feng et al. (1992) observed a special kind of hyphal bodies occurring late in the season. The hyphal bodies were spherical and clearly distinguishable from the regular hyphal bodies. While the appearance of these spherical hyphal bodies increased late in the season Feng et al. (1992) suggested that this kind of hyphal body may function as an overwintering form in the life cycle of P. neoaphidis. Therefore, Feng et al (1992) concluded that P. neoaphidis survives the winter months in the form of hyphal bodies on plant substrates rather than in the soil.

For E. planchoniana Keller (1997a) observed thick walled hyphal bodies in populations of Drepanosiphum acerinum (Walker). The proportion of infected aphids developing thick walled hyphal bodies instead of conidial infections was shown to increase during fall. Keller (1997a) also showed that in spring it was possible to infect healthy aphids with E. planchoniana from aphids containing thick walled hyphal bodies.

4.4 Conidia and ’loriconidia’

Studies of conidial survival have primarily been concentrated on P. neoaphidis due to the lack of resting spores in this species. On fresh oilseed rape leaves Schofield et al. (1995) showed that conidia of P. neoaphidis remain infective of up to 32 days after incubation at 5^oC and 85% r.h. and only up to 16 days after exposure to winter field conditions. It is therefore unlikely that conidia on leaf surfaces are the only overwintering mechanism. Nevertheless, conidia left on the surface of the soil are probably able to remain infective for a longer period. On the surface of soil under wet and dark conditions Latteur & Randall (1986) documented that primary conidia were able to produce replicate conidia for 24 days at 20^oC and for 6 - 8 months at 5^oC. They concluded that this must be the way that P. neoaphidis survives during winter. Morgan (1994) found in agreement with Latteur & Randall (1986) that primary conidia of P. neoaphidis were able to produce secondary conidia for 16 days at 18^oC and for at least 8 months at 5^oC on soil kept at a water holding capacity of 50%. However, Morgan (1994) also ran the experiment at 10^oC and found that replicate conidia were only produced for one month. Morgan (1994) concluded that at least in Britain the winter temperature is not consistently low enough for the survival of P. neoaphidis as conidia. For C. obscurus it has been shown that conidia showered onto the surface of nonsterile soil can produce replicate conidia for several months and that these conidia can actually infect aphids (Latteur, 1980).

Weiser & Batko (1966) observed in their studies of Conidiobolus destruens (Weiser & Batko) Ben-Ze’ev & Kenneth thick walled external conidia and descibed them as 'loriconidia'. They suggested that this structure was an alternative way of winter survival for Entomophthoralean fungi.

To investigate the survival structure of P. neoaphidis we incubated non-sporulating cadavers of S. avenae on sterilised soil in darkness at 5^oC. After one month we examined the cadavers. In addition we examined conidia produced in vitro on solid media after approximately three months of storage. Around the stored cadavers we observed conidia similar to those Weiser & Batko (1966) earlier described as 'loriconidia' for C. destruens. From the stored in vitro cultures, thick walled conidia were observed as well. Thick walled hyphal bodies were not observed in this study.

4.5 Anholocyclic aphid populations

Finally it has been suggested that P. neoaphidis survives in anholocyclic aphid populations via continuous conidial infections (Byford and Reeves, 1969; Wilding, 1973). Byford and Reeves (1969) found in spring that the peach-potato aphid, Myzus persicae Sulz. and the mangold aphid, Rhopalosiphoninus staphylae Koch, were infected with P. neoaphidis at their overwintering place, beet clamps. They concluded that P. neoaphidis was able to survive on aphids in clamps prior to dispersal in spring.

In anholocyclic aphid populations in northern littoral France P. neoaphidis has been observed throughout the year even during winter (Remaudière et al., 1981). However winter temperatures are much higher there than in Denmark.

4.6 Soil as environment for survival of Pandora neoaphidis

Objective

The environment for survival, independant of the survival structure, may be either soil, leaves, trunks or in anholocyclic aphid populations. In this study we surveyed soil as a natural source of inoculum for Entomophthorales infecting aphids, particularly with reference to the winter survival of P. neoaphidis.

During 1997, 1998 and 1999 soils were sampled before immigration of aphids to their summer hosts. Soil from the surface was sampled underneath bird cherry in an organically grown beet field with winter wheat as the previous crop, and in permanent grass. Bioassays were conducted to evaluate infection of S. avenae by Entomophthoralean fungi in the soil samples. For each bioassay between 40 and 50 petri-dishes with soil samples were used for screening of fungi once or twice per week for four weeks. Between each bioassay soil samples were incubated at 17^oC and 12:12 L:D, until the next bioassay began. For each bioassay twelve 3rd-4th instar S. avenae nymphs were placed on the soil samples for 18 hours and then transferred to winter wheat seedlings and incubated at 20^oC. The aphids were monitored daily for one week to detect infection with entomophthoralean fungi (for more detailed information concerning materials and methods see Appendix C).

The percentage of soil samples (data from all localities are pooled) containing inoculum, as evidenced by S. avenae infection, is shown as a function of days after sampling in table 4.3. This study documented that P. neoaphidis and C. obscurus were present in soil from different ecosystems prior to immigration of cereal aphids in spring. Since the aphids were not always infected immediately following sampling of the soil, it is likely that the inoculum is dormant or quiescent during the winter. The breaking of dormancy or quiescence is thought to be a very complex process controlled by the fungus species as well as by temperature and humidity.

Table 4.3
Entomophthoralean fungi on soil surfaces as evidenced by Sitobion avenae infection after 18 to 24 hours of contact with the soil. The percentages of soil samples which caused infection with entomophthoralean fungi are shown as a function of time. The soil samples were incubated at 17^oC and 12:12 L:D in 1997 and 14:10 L:D in 1998 and 1999.