Entomopathogenic Fungus Pandora Neoaphidis Research Articles

Computer-assisted image processing to detect spores from the fungus Pandora neoaphidis

This contribution demonstrates an example of experimental automatic image analysis to detect spores prepared on microscope slides derived from trapping. The application is to monitor aerial spore counts of the entomopathogenic fungus Pandora neoaphidis which may serve as a biological control agent for aphids. Automatic detection of such spores can therefore play a role in plant protection. The present approach for such detection is a modification of traditional manual microscopy of prepared slides, where autonomous image recording precedes computerised image analysis. The purpose of the present image analysis is to support human visual inspection of imagery data – not to replace it. The workflow has three components:•Preparation of slides for microscopy.•Image recording.•Computerised image processing where the initial part is, as usual, segmentation depending on the actual data product. Then comes identification of blobs, calculation of principal axes of blobs, symmetry operations and projection on a three parameter egg shape space.

Mesocosm experiments to assess the transmission of Pandora neoaphidis within simple and mixed field margins and over the crop-margin interface

Although considerable research on the development of agri-environment schemes has focussed on the value of managed field margins as reservoirs for arthropod natural enemies, their potential as reservoirs of entomopathogenic fungi has received less attention. Whether field margins that are most beneficial for arthropod natural enemies are the same as those for entomopathogenic fungi is unknown. Here, within glasshouse mesocosms, we assessed the reproductive success of the aphid-specific entomopathogenic fungus Pandora neoaphidis on aphids in a ‘simple margin’ containing one plant species and on the same species of aphid in a ‘mixed margin’ containing seven plant species. These assessments were done in the presence of Aphidius ervi, a hymenopteran parasitoid of aphids regarded as being a key species to conserve in agri-environment schemes in the UK. When only the plants initially infested with aphids were assessed, transmission of P. neoaphidis was significantly greater (p<0.001) in the mixed margin as was parasitisation by A. ervi (p<0.05). However, when all of the plants in the mesocosms were assessed, transmission of P. neoaphidis remained greater in the mixed margin (p<0.05) whereas parasitisation by A. ervi was greater in the simple margin (p<0.05). This difference may be due to aphid dispersal which was greater in the simple margin thereby benefitting the actively foraging parasitoid whereas clustering of aphids in the mixed margin benefited the passively dispersed fungus. In a second mesocosm experiment, the movement of P. neoaphidis over the crop-margin interface was similar to that of A. ervi despite the fungus only being passively dispersed in contrast to the actively foraging parasitoid. The results presented here indicate that, although the optimal plant composition of field margins may differ for P. neoaphidis and A. ervi, both species can co-exist and reproduce in field margins and will move over the crop-margin interface. Managed field margins that benefit both key arthropod and key microbial enemies have potential for enhancing pest control in associated crops.

First report of Pandora neoaphidis resting spore formation in vivo in aphid hosts

The entomopathogenic fungus Pandora neoaphidis is a recognized pathogen of aphids, causes natural epizootics in aphid populations, and interacts and competes with aphid predators and parasitoids. Survival of entomophthoralean fungi in periods of unsuitable weather conditions or lack of appropriate host insects is accomplished mainly by thick-walled resting spores (zygospores or azygospores). However, resting spores are not known for some entomophthoralean species such as P. neoaphidis. Several hypotheses of P. neoaphidis winter survival can be found in the literature but so far these hypotheses do not include the presence of resting spores. Resting spores were found in an aphid population where P. neoaphidis was the only entomophthoralean fungus observed during surveys conducted in organic horticultural crops in greenhouses and open fields in Buenos Aires province, Argentina. This study sought to use molecular methods to confirm that these resting spores were, in fact, those of P. neoaphidis while further documenting and characterizing these resting spores that were produced in vivo in aphid hosts. The double-walled resting spores were characterized using light and transmission electron microscopy. The Argentinean resting spores clustered together with P. neoaphidis isolates with bootstrap values above 98 % in the small subunit ribosomal RNA (SSU rRNA) sequence analysis and with bootstrap values above 99 % the Internal Transcribed Spacer (ITS) II region sequence analysis. This study is the first gene-based confirmation from either infected hosts or cultures that P. neoaphidis is able to produce resting spores.

The effect of the coccinellid Harmonia axyridis (Coleoptera: Coccinellidae) on transmission of the fungal pathogen Pandora neoaphidis (Entomophthorales: Entomophthoraceae)

The coccinellid Harmonia axyridis is a recent arrival in the UK and is an intraguild predator of the entomopathogenic fungus Pandora neoaphidis. Harmonia axyridis entirely consumes P. neoaphidis-sporulating cadavers and this may have a negative effect on the epizootic potential of P. neoaphidis. Here we assessed within plant transmission, and between plant vectoring, of P. neoaphidis in the presence of either H. axyridis or Coccinella septempunctata, a native coccinellid that only partially consumes fungal cadavers. Transmission was greater in the presence of coccinellids, with 21% of aphids becoming infected with the fungus whilst only 4% were infected in the control. However, there was no significant effect of coccinellid species or sex on fungal transmission. Between plant vectoring occurred infrequently in the presence of both species of coccinellid. The effect of H. axyridis on P. neoaphidis transmission is, therefore, likely to be similar to that of the native coccinellid C. septempunctata.

Potential value of the fibre nettle Urtica dioica as a resource for the nettle aphid Microlophium carnosum and its insect and fungal natural enemies

The perennial stinging nettle (Urtica dioica L.) is a wild plant that provides resources for aphid natural enemies and can therefore benefit crop protection. Stinging nettles producing approximately three times more fibre than Standard nettles are under commercial development for fibre production. Here we assess the relative value of Austrian Clone 2, a high fibre nettle variety, as a resource for the nettle aphid Microlophium carnosum (Buckton) and its associated natural enemies. The intrinsic rate of increase of M. carnosum cultured on Clone 2 was not different to that on a Standard nettle nor was there an effect of nettle variety on the susceptibility of this aphid to the entomopathogenic fungus Pandora neoaphidis (Remaudiere and Hennebert) Humber. The development time of the aphidophagous predator Chrysoperla carnea (Stephens) was not affected by the nettle variety on which M. carnosum was cultured whilst the parasitoid Aphidius ervi (Haliday) fed on honeydew produced by M. carnosum infesting both varieties of nettle. Fibre nettle Clone 2 is therefore able to support non-pest aphids and their natural enemies and, if grown widely in the future, may be useful within conservation biological control and a tool within integrated pest management.

BIOLOGICAL CHARACTERISTICS OF SLOVAK ISOLATES OF ENTOMOPATHOGENIC FUNGUS PANDORA NEOAPHIDIS (REMAUDIÈRE ET HENNEBERT) HUMBER (ZYGOMYCETES, ENTOMOPHTHORALES)

Intraspecific variability of biological characteristics within entomopathogenic fungus Pandora neoaphidis was evaluated. Fifteen isolates of the fungus were obtained from 5 aphid species in Slovakia. Size of conidia, conidial germination, virulence, radial growth, and biomass production were evaluated. Conidial size varied considerably with exception of isolates originating from the same host population. Conidial germination was observed on all the surfaces tested and it was greatest at saturated humidity. Virulence, daily rate of radial growth and biomass production varied depending on isolates. Isolates obtained from the same host colonies during fungal epizootics shoved also significant differences in the characteristics, what may suggest that epizootics in aphid populations are caused by associations of strains and not by prevalence of a single virulent strain.

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.

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.

Influence of the aphid pathogen Pandora neoaphidis on the foraging behaviour of the aphid parasitoid Aphidius ervi

Abstract. 1. The parasitoid Aphidius ervi and the entomopathogenic fungus Pandora neoaphidis both require successful invasion of an aphid host to complete their life cycle. A shorter developmental period allows P. neoaphidis to out‐compete A. ervi. Aphidius ervi may reduce this fitness cost by avoiding aphid colonies containing P. neoaphidis. Here the response of A. ervi towards P. neoaphidis was assessed using sequential experiments designed to replicate different stages of parasitoid foraging behaviour.2. Entry rate experiments showed that A. ervi entered aphid colonies containing P. neoaphidis‐sporulating cadavers and that there was no significant difference in the attraction of A. ervi to aphid‐damaged Vicia faba plants containing either healthy Acyrthosiphon pisum or P. neoaphidis‐sporulating cadavers.3. Observational behavioural experiments indicated that the presence of P. neoaphidis did not affect the search time or total foraging time of A. ervi on V. faba plants infested with either healthy A. pisum or P. neoaphidis‐sporulating cadavers.4. In Petri dish bioassays using aphids infected with P. neoaphidis over a period of 120 h, A. ervi showed no difference in attack rate against uninfected aphids or living aphids infected with P. neoaphidis for 1, 24, 48, 72, or 96 h. However, sporulating cadavers (120 h infection) were not attacked.5. Aphidius ervi appears only able to detect the presence of P. neoaphidis once the host is dead and sporulation has started. The fitness of A. ervi may therefore be severely reduced when foraging in P. neoaphidis‐infected aphid colonies.

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.

ISSR, ERIC and RAPD techniques to detect genetic diversity in the aphid pathogen Pandora neoaphidis

The entomopathogenic fungus Pandora neoaphidis is an important natural enemy of aphids. ISSR, ERIC (Enterobacterial Repetitive Intergenic Consensus) and RAPD PCR-based DNA fingerprint analyses were undertaken to study intra-specific variation amongst 30 isolates of P. neoaphidis worldwide, together with six closely related species of Entomophthorales. All methods yielded scorable binary characters, and distance matrices were constructed from both individual and combined data sets. Neighbour-joining was used to construct consensus phylogenetic trees which showed that although P. neoaphidis isolates were highly polymorphic they separated into a monophyletic group compared with the other Entomophthorales tested. Three distinct subclades were found, with UK isolates occupying two of these. No specific correlation with aphid host species was established for any of the isolates apart from those in one cluster which contained isolates obtained from nettle aphid, Microlophium carnosum. ERIC, ISSR and RAPD analysis allowed the rapid genetic characterisation and differentiation of isolates with the generation of potential isolate- and cluster specific-diagnostic DNA markers.

Direct and indirect effects of wheat cultivars with different levels of resistance on parasitoids and entomopathogenic fungi of cereal aphids

Laboratory experiments were performed to evaluate at the population level the influence of host-plant resistance on interspecific interactions of the cereal aphid Sitobion avenae with two phylogenetically distant species of natural enemies, the parasitoid Aphidius rhopalosiphi and the entomopathogenic fungus Pandora neoaphidis. Indirect effects of wheat resistance on interspecific interactions between parasitoid and fungus were caused by interaction modifications, i.e., changes in per capita interspecific effects, as evidenced by significant statistical interactions between the effects of the fungus and wheat resistance on per capita population growth rate (PCPGR) of the parasitoid. Reduction by the fungus of parasitoid PCPGR was lower on the susceptible wheat than on the resistant wheat; this was interpreted as the population level expression of the asymmetric competitive effect of the fungus on parasitoid development. As expected from the asymmetric interaction between parasitoid and fungus, no indirect effect of the parasitoid on the fungus was found, as evidenced by no significant statistical interactions between wheat cultivar and parasitoid. However, increased fungus PCPGR was found in the presence of the parasitoids regardless of wheat cultivar; this may also be explained by increased fungal infection during parasitoid foraging. Path analysis, a statistical technique to discern the relative contributions of direct and indirect effects, also detected indirect effects between fungus and parasitoid.

Effect of wheat resistance, the parasitoid Aphidius rhopalosiphi, and the entomopathogenic fungus Pandora neoaphidis, on population dynamics of the cereal aphid Sitobion avenae

AbstractThe influence of wheat (Triticum aestivum L.) resistance, the parasitoid Aphidius rhopalosiphi De Stephani‐Perez (Hymenoptera: Braconidae) and the entomopathogenic fungus Pandora neoaphidis (Remaudière et Hennebert) Humber (Zygomycetes: Entomophthorales) on the density and population growth rate of the cereal aphid Sitobion avenae (F.) (Hemiptera: Aphididae) was studied under laboratory conditions. Partial wheat resistance was based on hydroxamic acids, a family of secondary metabolites characteristic of several cultivated cereals. The partial resistance of wheat cultivar Naofén, the action of the parasitoid and the joint action of the parasitoid and fungus, reduced aphid density. The lowest aphid densities were obtained with the combination of the parasitoid and the fungus, but wheat resistance under these circumstances did not improve aphid control. Significant reductions of population growth rate (PGR) of aphids were obtained with the joint action of wheat resistance and natural enemies. In particular, the combined effects of parasitoids and fungi showed significantly lower PGR than the control without natural enemies in both wheat cultivars. Our results support the hypothesis that wheat resistance and the utilization of biological control agents could be complementary strategies in an integrated pest management program against cereal aphids.

Green Leaf Volatiles Inhibit Conidial Germination of the Entomopathogen Pandora neoaphidis (Entomopthorales: Entomophthoraceae)

We tested whether a tobacco, Nicotiana tabacum (L.), plant defense against tobacco aphids, Myzus nictianae (Blackman), might also inhibit the entomopathogenic fungus Pandora neoaphidis (Remaudiere & Hennebert). Growth of fungal colonies exposed to macerated and unmacerated tobacco leaves, hexanal, (E)-2-hexenal, and (E)-2-hexenol was strongly suppressed compared with controls. Large numbers of ungerminated conidia were recovered from the media surrounding the treated colonies, whereas few ungerminated conidia were found in media surrounding the controls. Conidia were then collected onto glass coverslips and exposed to the same treatments during the first 4 h after conidial relase. Relative to controls, hexanal substantially suppressed conidial germination, the leaf material suppressed germination substantially more than hexanal and (E)-2-hexenal was the strongest inhibitor indicating that the principal effect of the plant volatiles was inhibition of conidial germination. Based on scanning electron microscope examination, when conidia were deposited directly onto live aphids instead of cover slips, these volatiles had no effect on aphid infection rates, indicating that the inhibition of conidial germination does not occur when conidia are actually on an aphid surface. Finally, germination of conidia exposed to volatiles of plants previously infested with aphids was substantially suppressed relative to uninfested controls. These studies suggest that tobacco plant volatiles produced in response to wounding such as aphid feeding inhibit conidial germination by the entomopathogen unless the conidia are in direct contact with the aphid integument. We suggest that the conidia respond to presence of wound-induced volatiles by delaying germination so as to increase their probability of finding an aphid host ; the conidia use the plant's wound response as a cue to indicate host proximity. A conceptual model of this process is presented.