Pandora neoaphidis transmission and aphid foraging behaviour

Pandora neoaphidis is an aphid-specific entomopathogen that produces infective conidia. As aphid movement increases, so does the likelihood of contact with conidia. Volatile distress signals released in response to aphid infestation as an indirect defence against herbivory may affect aphid foraging and, therefore, the fungus-aphid interaction. In this study, two different methods were used to investigate the effect of plant volatiles and P. neoaphidis-sporulating cadavers on (1) the colonisation of Vicia faba plants by Acyrthosiphon pisum and (2) P. neoaphidis transmission. This study indicates that A. pisum does not avoid bean plants containing P. neoaphidis and that transmission of conidia occurs during plant colonisation and, to a lesser extent, during in situ feeding. Although significantly more aphids were recovered from damaged plants compared to undamaged plants, the likelihood of infection was not affected by previous infestation by aphids.     

A PCR-based tool for the cultivation-independent monitoring of Pandora neoaphidis

Pandora neoaphidis is one of the most important fungal pathogens of aphids and has a great potential for use in biocontrol. Little is known on how this fungus persists in an area and in particular on its overwintering strategies. It is hypothesized that natural areas play an important role for survival and that soil may serve as a source of inoculum for new aphid populations in spring. To test these hypotheses, a cultivation-independent PCR-based diagnostic tool was developed, that allows the detection of P. neoaphidis in the environment. Two P. neoaphidis specific PCR primer pairs were designed, targeting sequences in the ribosomal RNA gene cluster. Specificity of both primer pairs was demonstrated with P. neoaphidis and non-target close entomophthoralean relatives. Moreover, single amplicons of expected sizes were obtained with both primer pairs from various environmental sample types, including aphid cadavers, plant material, and soil. The PCR-based diagnostic tool was applied to investigate the persistence of P. neoaphidis in soil samples obtained in 2004/2005 from a nettle field harboring infected aphids in fall 2004. P. neoaphidis was detected in every sample collected in November 2004 and March 2005, suggesting an overwintering stage of P. neoaphidis in top soil layers. The developed cultivation-independent PCR-based tool will be valuable for further investigation of the ecology of P. neoaphidis and for the development and future implementation of management strategies against aphids involving conservation biocontrol.     

Transmission of Pandora neoaphidis in the presence of co-occurring arthropods

Transmission of the entomopathogenic fungus Pandora neoaphidis to the nettle aphid Microlophium carnosum was assessed in the presence of arthropods that co-exist with the fungus within the habitat but do not compete for aphid hosts. The presence of a parasitoid significantly enhanced transmission, and transmission rates were similar for both enemy and non-enemy parasitoids. Although herbivory of nettle leaves by Peacock butterfly (Inchis io) caterpillars indirectly reduced the number of M. carnosum by >30% due to a reduction in leaf area for feeding, the addition of I. io significantly increased transmission of P. neoaphidis in the remaining aphids. It is likely that enhanced transmission in the presence of A. rhopalosiphii and I. io is due to disturbance and subsequent movement of the aphid, resulting in contact with conidia deposited on the leaf surface. The presence and impact of co-occurring arthropods should be taken into consideration when assessing the transmission of fungal entomopathogens.     

Response of the entomopathogenic fungus Pandora neoaphidis to aphid-induced plant volatiles

We used a model plant-aphid system to investigate whether the aphid-specific entomopathogenic fungus Pandora neoaphidis responds to aphid-induced defence by the broad-bean plant, Vicia faba. Laboratory experiments indicated that neither in vivo sporulation, conidia size nor the in vitro growth of P. neoaphidis was affected by Acyrthosiphon pisum-induced V. faba volatiles. The proportion of conidia germinating on A. pisum feeding on previously damaged plants was significantly greater than on aphids feeding on undamaged plants, suggesting a direct functional effect of the plant volatiles on the fungus. However, there were no significant differences in the infectivity of P. neoaphidis towards A. pisum feeding on either undamaged V. faba plants or plants previously infested with A. pisum. Therefore, these results provide no evidence to suggest that P. neoaphidis contributes to plant indirect defence strategies.     

Effect of seasonal abiotic conditions and field margin habitat on the activity of Pandora neoaphidis inoculum on soil

The ability of the aphid pathogenic fungus Pandora neoaphidis to remain active in the absence of a resting stage through a combination of continuous infection and as conidia deposited on soil was assessed alongside the potential for planted field margins to act as a refuge for the fungus. P. neoaphidis was able to infect the pea aphid, Acyrthosiphon pisum, when maintained under controlled conditions that simulated those that occur seasonally in the UK. Although there was a significant inverse relationship between temperature and time-to-kill, with death occurring after 4.2, 6.9 and 13.6 days when maintained under fluctuating summer, autumn and winter temperatures, respectively, there were no additional statistically significant effects of photoperiod. The activity of inoculum on soil was indirectly assessed by baiting with A. pisum. Under controlled conditions P. neoaphidis remained active on soil and was able to infect aphids for up to 80 days. However, the percentage of aphids that became infected decreased from 76% on day 1 to 11% on day 80. Whereas there was little difference in the activity of conidia that had been maintained at 4 degrees C and 10 degrees C, activity at 18 degrees C was considerably reduced. Under field conditions the activity of inoculum was strongly influenced by season. On day 49 there was little or no activity during spring, summer or winter. However, during autumn a mean proportion of 0.08 aphids still became infected with P. neoaphidis. Margin type did not affect the activity of conidia nor was there a difference in activity between blocks that had regenerated naturally and those that had been planted. These results suggest that P. neoaphidis can infect aphids and remain active on soil under the abiotic conditions that occur seasonally in the UK and that this fungus may be able to persist annually without a resting stage.     

Plant waxy bloom on peas affects infection of pea aphids by Pandora neoaphidis

This study examined the effects of the surface wax bloom of pea plants, Pisum sativum, on infection of pea aphids, Acyrthosiphon pisum, by the fungal pathogen Pandora neoaphidis. In prior field surveys, a higher proportion of P. neoaphidis-killed pea aphids (cadavers) had been observed on a pea line with reduced wax bloom, as compared with a sister line with normal surface wax bloom. Laboratory bioassays were conducted in order to examine the mechanisms. After plants of each line infested with aphids were exposed to similar densities of conidia, the rate of accumulation of cadavers on the reduced wax line was significantly greater than on the normal wax bloom line; at the end of the experiment (13d), the proportion of aphid cadavers on the reduced wax line was approximately four times that on the normal wax bloom line. When plants were exposed to conidia first and then infested with aphids, the rate of accumulation of cadavers was slightly but significantly greater on the reduced wax line, and infection at the end of the experiment (16d) did not differ between the lines. When aphids were exposed first and then released onto the plants, no differences in the proportion of aphid cadavers were observed between the pea lines. Greater infection of pea aphid on reduced wax peas appears to depend upon plants being exposed to inoculum while aphids are settled in typical feeding positions on the plant. Additional experiments demonstrated increased adhesion and germination by P. neoaphidis conidia to leaf surfaces of the reduced wax line as compared with normal wax line, and this could help explain the higher infection rate by P. neoaphidis on the reduced wax line. In bioassays using surface waxes extracted from the two lines, there was no effect of wax source on germination of P. neoaphidis conidia.     

Sitobion avenae alatae infected by Pandora neoaphidis: their flight ability, post-flight colonization, and mycosis transmission to progeny colonies

Epizootics caused by the obligate Entomophthorales pathogen Pandora neoaphidis may result from more than one possible means of fungal dissemination among host aphids, but we hypothesize that wide dispersal of the fungus is most likely to be associated with the flight behavior of migratory alates. We tested this hypothesis in a simulation experiment by assessing the flight capability of Sitobion avenae alates infected with P. neoaphidis and the potential of their post-flight survival, colonization, and mycosis transmission to progeny. A total of 281 alates were inoculated with P. neoaphidis, individually flown for up to 5h and 9km in a computer-monitored flight mill system and then reared for 10 days on wheat seedlings. The infected alates were capable of surviving on average for 2.9 days (range 1-7 days) and leaving 4.6 nymphs prior to deaths. Transmission of fungal infection within progeny colonies occurred after the mother alates died from P. neoaphidis mycosis. The level of contagious infection among the nymphs reached up to 16.8% within 7 days but varied with the survival time of the infected mother alates after flight. Based on stepwise polynomial regression analysis, progeny colony size was highly correlated with the interactions of flight time with both post-flight survival time and the number of nymphs left per alate before death (r2 = 0.997). Progeny mortality on day 5 after colonization was inversely correlated with post-flight survival time (r2 = 0.949) whereas infection on day 7 was correlated with flight distance and an interaction of post-flight survival time with fecundity of the infected alates (r2 = 0.970). Progeny mortality observed on day 10 was merely correlated to mortality observed on day 5 (r2 = 0.946). These results indicate a successful transmission of alate-borne P. neoaphidis to progeny colonies and further support our hypothesis on the means of primary dispersal of aphid epizootics by migratory alates in a geographically wide range.     

Biotic and abiotic regulation of resting spore formation in vivo of obligate aphid pathogen Pandora nouryi: Modeling analysis and biological implication

Entomophthoralean fungus Pandora nouryi is an obligate aphid pathogen that enables to produce resting spores (azygospores) for surviving host absence. To explore possible mechanisms involved in the regulation of resting spore formation in vivo, host cohorts consisting of 40-60 nymphs of green peach aphid Myzus persicae produced within 24 h on cabbage leaf discs in petri dishes were exposed to spore showers of P. nouryi at the concentrations (C) from a very few to nearly 2000 conidia/mm² and then reared for 7-11 days at the regimes of 10-25 °C (T) and 8-16 h daylight (H_L) or ambient (17.5 ± 3.1 °C, 13:11 L:D). Aphid mortalities observed from 35-83 cohorts (showered separately) at each regime showed typical sigmoid trend and fit well a general logistic equation (0.79 ? r² ? 0.88), yielding similar LC₅₀ estimates of 1.7-6.1 conidia/mm². The proportions (P) of cadavers forming resting spores in the cohorts also fit the same equation (0.73 ? r² ? 0.85) at all tested regimes except at 10 °C, a low temperature for the host-pathogen interaction. This indicates the dependence of resting spore formation on the spore concentration. The effects of T and H_L on P over C were well elucidated by the fitted modified logistic equations P = 0.578/{1 + exp[1.710 − (0.136 − 0.0053T)C]} and P = 0.534/{1 + exp[1.639 + (0.034 − 0.0053H_L)C]} (both r² = 0.79). Our results highlight that the resting spore formation in vivo of P. nouryi is regulated primarily by the concentration of host-infecting conidia discharged from cadavers and facilitated by lower temperature and longer daylight.     

Intraguild interactions between the entomopathogenic fungus Pandora neoaphidis and an aphid predator and parasitoid at the population scale

The interactions that occur between the entomopathogenic fungus Pandora neoaphidis and a predator (Coccinella septempunctata) and a parasitoid (Aphidius ervi) were assessed in microcosm and polytunnel experiments. Transmission of P. neoaphidis to the pea aphid, Acyrthosiphon pisum, was enhanced in the presence of both C. septempunctata and A. ervi in microcosm experiments done under fixed abiotic conditions. In contrast, the reproductive success of A. ervi was reduced in the presence of P. neoaphidis. Despite the increased fungal transmission in the presence of C. septempunctata, there was no additional decrease in the aphid population indicating that P. neoaphidis is functionally redundant in the presence of the coccinellid. In polytunnel experiments the reproductive success of A. ervi was not affected by P. neoaphidis. These results do not support those of the microcosm and may be due to the more natural abiotic conditions in the polytunnel reducing the competitive advantage of the fungus. Microcosms therefore provide an arena in which the interactions between fungal pathogens and other aphid-natural enemies can be assessed however, further assessments at increased spatial scales under more natural abiotic conditions are also required to accurately determine the outcome of these interactions.     

Effects of 20 fungicides on the infectivity of conidia of the aphid entomopathogenic fungus Erynia neoaphidis

The effect of 20 fungicides on theinfectivity of conidia of the entomopathogenicfungus, Erynia neoaphidis, were assessedin the laboratory. After projection on broadbean leaves, conidia were treated withfungicides applied at their recommended fieldrate. Afterwards, the infectivity of theseinocula was assessed using an aphid bioassay.Four fungicides, carbendazim, kresoxym-methyl, nuarimol and thiophanate-methyl reduced the infectivity of the conidia by less than 25% and can be considered harmless for this aphid pathogen. Propiconazole was a little more toxic, with 37% reduction. Other products reducedinfectivity by between 50% and 100%. These are, from the least to the most toxic:flutriafol, prochloraz, epoxyconazole,iprodione, hexaconazole, triadimenol,azoxystrobine, cyproconazole, cyprodynil,flusilazole and tridemorph. Chlorothalonil,fenpropimorph, spiroxamine and tebuconazoletotally inhibited infectivity of the fungi. Analysis of the results according to chemicalclass showed that the benzimidazoles were theleast toxic for E. neoaphidis and themorpholines the most toxic. Effects oftriazoles and strobilurines were variable, withreduction ranging from 37% to 100% fortriazoles and from 17% to 68% forstrobilurines.     

Altered behavior and distribution of pea aphids, Acyrthosiphon pisum (Homoptera: Aphididae), infected with Pandora neoaphidis (Zygomycetes: Entomophthorales)

The distribution and mobility of infected aphid hosts can have a great effect on the ability of a pathogen to spread throughout a population. The distribution of dead and living pea aphids (Acyrthosiphon pisum) infected with Pandora neoaphidis was compared with that of their healthy conspecifics. Infected aphids were significantly more likely to be found on the undersides of alfalfa leaves and off of the plants than were healthy aphids. These two shifts in microhabitat location have potential costs and benefits for both the host and the pathogen.     

Non-target impacts of soybean rust fungicides on the fungal entomopathogens of soybean aphid

Soybean aphid, Aphis glycines, has caused serious economic damage to soybean across the North Central US since its introduction to North America in 2000. The management of another invasive soybean pest, Asian soybean rust, Phakopsora pachyrhizi, using foliar fungicide applications has the potential to impact soybean aphid populations by suppressing beneficial fungal entomopathogens. In 2005 and 2006, we applied recommended soybean rust fungicide treatments, consisting of strobilurin and triazole fungicides, to small soybean plots in two locations to assess if such applications might suppress aphid fungal epizootics. In Lamberton, MN, in 2005, during the epizootic, fungicide-treated plots averaged 2.0 ± 0.7% (mean ± SE) disease prevalence while untreated plots averaged 14.2 ± 5.6%. In 2007, we applied strobilurin and strobilurin-triazole mix fungicides to single-plant microplots either before or after release of Pandora neoaphidis, the most commonly observed aphid pathogen in 2005 and 2006. Treatments that contained a mixture of two active ingredients significantly lowered peak and cumulative aphid disease prevalence in both early and late reproductive stage soybeans indicating that fungicide mixtures used to manage soybean rust can negatively impact an aphid-specific fungal pathogen. However, no consistent soybean aphid population response was observed in these studies of low levels of aphid fungal infection.     

Molecular-genetic evidence of aphid pathogen Pandora kondoiensis infection in Sitobion avenae collected in Denmark

Entomophthoralean-specific primers were used to estimate the occurrence of entomophthoralean species in the cereal aphid Sitobion avenae in Denmark. Sequencing documented for the first time the presence of Pandora kondoiensis in Denmark. Specific primers were designed and P. kondoiensis was identified on 12% of infested straws sampled in 2012.     

Ultrastructure of mitosis in the aphid-pathogenic fungusErynia neoaphidis

Summary An account of mitosis in the aphid-pathogenic, entomophthoraceous fungusErynia neoaphidis is presented. The mitotic apparatus is characterized by a closed, intranuclear, polarized spindle. Chromosomes are permanently attached by kinetochore microtubules (kcMTs) to the poles during mitosis. The spindle develops as the spindle pole bodies migrate and separate. At metaphase the eccentric spindle contains only kcMTs and is located in a relatively chromatinfree zone. Paired sister kinetochores are arranged in a broad metaphase plate. During anaphase kcMTs shorten, astral and nonchromosomal microtubules develop and elongate and the interpolar distance increases.     

Effect of fungal infection on the reproductive potential of aphids and their progeny

The effect of infection by Pandora neoaphidis and Beauveria bassiana on the reproductive potential of the pea aphid, Acyrthosiphon pisum, and their progeny was assessed. Infection by either P. neoaphidis or B. bassiana reduced the number of nymphs produced within 24 h of inoculation and over the entire infection period compared to uninfected aphids. However, infection by either P. neoaphidis or B. bassiana for 24 or 72 h did not alter the intrinsic rate of increase of the host aphid's progeny. Therefore, fungal infection appears to have no indirect effects on the fitness of the host's progeny.     

Wheat containing snowdrop lectin (GNA) does not affect infection of the cereal aphid <Emphasis Type="Italic">Metopolophium</Emphasis><Emphasis Type="Italic">dirhodum</Emphasis> by the fungal natural enemy<Emphasis Type="Italic">Pandora neoaphidis</Emphasis>

Studies were carried out to determine if susceptibility of the cereal aphid Metopolophium dirhodum to the fungus Pandora neoaphidis was affected by wheat expressing snowdrop lectin (GNA). Aphid infection did not differ significantly between the transgenic GNA and non-transformed lines (91 and 82%, respectively). Fecundity also did not differ between aphids on the two lines, and was ca. 18 nymphs adult⁻¹. Time to infection was ca. 5 days for M. dirhodum on both lines in two of three assays. Our results indicate that wheat expressing GNA would not compromise the efficacy of P. neoaphidis as a biocontrol agent.     

The role of resting spores in the survival of the mite-pathogenic fungus Neozygites floridana from Mononychellus tanajoa during dry periods in Brazil

Survival of pathogens during long periods of unfavorable conditions can be critical to their ecology and to their use in biological control. In northeastern Brazil, the mite pathogen Neozygites floridana must survive hot and dry conditions between wet seasons when it infects the cassava green mite Mononychellus tanajoa. We report on large numbers of mite cadavers bearing resting spores towards the end of epizootics in mid-1995. High within-leaf variability indicated that local factors may be important in determining resting spore formation. These spores remain in the host cadaver on a leaf until the cadaver breaks up, whereupon the spores fall freely to the soil, there to remain dormant. Laboratory simulation of field conditions led to ca. 25% of mycosed individuals bearing resting spores. Mummies (without resting spores) kept in hot and dry conditions showed little or no viability within 2 months, implying no role for survival over extended dry periods. It is proposed that resting spores form the principal means by which this pathogen survives the dry season in the study area. This has implications for its introduction to new areas in classical biological control.     

First detection of the microsporidium Enterocytozoon bieneusi in non-mammalian hosts (chickens)

Faecal samples taken from eight underweight, approximately 5-week-old broiler chickens in a poultry abattoir were investigated for microsporidial infections by light microscopy, electron microscopy, and PCR. In two of six chickens, which were suspected of being infected with microsporidia by light microscopy, Enterocytozoon bieneusi (genotype 'J') was detected by PCR and DNA sequencing, and in one of the two PCR-positive samples by extensive electron microscopy. This is the first time that E. bieneusi has been detected in chickens, i.e. in a non-mammalian species.     

First record of Clonostachys rosea (Ascomycota: Hypocreales) as an entomopathogenic fungus of Oncometopia tucumana and Sonesimia grossa (Hemiptera: Cicadellidae) in Argentina

Clonostachys rosea (Link: Fries) Schroers, Samuels, Seifert, and Gams (Ascomycota: Hypocreales) has been reported as a mycoparasite of fungi and nematodes and as saprobe in soils, but this fungus has not been reported previously to be entomopathogenic. Many species of cicadellid leafhoppers cause economic damage to crops as vectors of plant pathogens. In the present work, we report the first record of C. rosea as an entomopathogenic fungus of two leafhoppers pest, Oncometopia tucumana and Sonesimia grossa (Hemiptera: Cicadellidae), in Argentina and evaluate the pathogenicity of C. rosea against them.     

Mannitol production from glycerol by resting cells of Candida magnoliae

Production of mannitol from glycerol by resting cells of Candida magnoliae under aerobic condition was investigated. The resting cells were suspended in aqueous solution of glycerol in Erlenmeyer flasks and incubated on rotary shaker. The samples were analyzed by ion exclusion–HPLC equipped with refractive index and UV detector. The resting cells of C. magnoliae produced mannitol from fructose, sucrose and glycerol but not from glucose. Addition of yeast extract and/or potassium phosphate to the glycerol solution adversely affected its conversion to mannitol. The conversion of glycerol to mannitol was dependent on oxygen availability. Using resting cells, the yield of mannitol was as high as 45%. This is probably the first report of conversion of glycerol to mannitol by osmophilic yeast.