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.     

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.     

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.     

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.     

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.     

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.     

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.     

Extraordinary proliferation of microorganisms in aposymbiotic pea aphids,Acyrthosiphon pisum

Aposymbiotic pea aphids, which were deprived of their intracellular symbiotic bacterium, Buchnera, exhibit growth retardation and no fecundity. High performance liquid chromatographic (HPLC) analysis revealed that these aposymbiotic aphids, when reared on broad bean plants, accumulated a large amount of histamine. To assess the possibility of extraordinary proliferation of microorganisms other than Buchnera, we enumerated eubacteria and fungi in aphids using the real-time quantitative PCR method that targets genes encoding small-subunit rRNAs. The result showed that these microorganisms were extremely abundant in the aposymbiotic aphids reared on plants. Microbial communities in aposymbiotic aphids were further profiled by phylogenetic analysis of small-subunit rDNAs. Of 172 nonchimeric sequences of fungal 18S rDNAs, 138 (80.2%) belonged to the phylum Ascomycota. Among them, 21 clustered within a monophyletic group consisting of insect-pathogenic fungi and yeast-like symbionts of homopteran insects. Thirty-one (18.0%), two (1.2%), and one (0.6%) clones were clustered within the Basidiomycota, Zygomycota, and Oomycota, respectively. Of 167 nonchimeric sequences of eubacterial 16S rDNAs, 84 (50.3%) belonged to the gamma-subdivision of Proteobacteria to which most primary endosymbionts of insects and prolific histamine producers belong. Forty (24.0%), 25 (15.0%), 10 (6.0%), and five (3.0%) clones were clustered within alpha-Proteobacteria, Cytophaga-Flavobacterium-Bacteroides (CFB) group, Actinobacteria, and beta-Proteobacteria, respectively. Three had no phylogenetic association with known taxonomic divisions. None of the sequences studied in this study coincided exactly with those deposited in GenBank.     

Effects of bacterial secondary symbionts on host plant use in pea aphids

Aphids possess several facultative bacterial symbionts that have important effects on their hosts'' biology. These have been most closely studied in the pea aphid (Acyrthosiphon pisum), a species that feeds on multiple host plants. Whether secondary symbionts influence host plant utilization is unclear. We report the fitness consequences of introducing different strains of the symbiont Hamiltonella defensa into three aphid clones collected on Lathyrus pratensis that naturally lack symbionts, and of removing symbionts from 20 natural aphid–bacterial associations. Infection decreased fitness on Lathyrus but not on Vicia faba, a plant on which most pea aphids readily feed. This may explain the unusually low prevalence of symbionts in aphids collected on Lathyrus. There was no effect of presence of symbiont on performance of the aphids on the host plants of the clones from which the H. defensa strains were isolated. Removing the symbiont from natural aphid–bacterial associations led to an average approximate 20 per cent reduction in fecundity, both on the natural host plant and on V. faba, suggesting general rather than plant-species-specific effects of the symbiont. Throughout, we find significant genetic variation among aphid clones. The results provide no evidence that secondary symbionts have a major direct role in facilitating aphid utilization of particular host plant species.     

Comparative proteomic analysis of BTH and BABA-induced resistance in pea (Pisum sativum) toward infection with pea rust (Uromyces pisi)

Systemic acquired resistance (SAR) to Uromyces pisi in pea was studied by using a proteomic approach. Two-dimensional electrophoresis (2-DE) was used in order to compare the leaf proteome of two pea genotypes displaying different phenotypes (susceptible and partial resistance to the fungus), and in response to parasite infection under the effect of two inducers of SAR, BTH and BABA. Multivariate statistical analysis identified 126 differential protein spots under the experimental conditions (genotypes/treatments). All of these 126 protein spots were subjected to MALDI-TOF/TOF mass spectrometry to deduce their possible functions. A total of 50 proteins were identified using a combination of peptide mass fingerprinting (PMF) and MSMS fragmentation. Most of the identified proteins corresponded to enzymes belonging to photosynthesis, metabolism, biosynthesis, binding and defense response, whose behavior pattern was different in relation to susceptibility/resistance of the genotypes studied and to the BTH/BABA induction to pathogen response. Results obtained in this work suggested that plants could reduce their photosynthesis and other energy metabolism and enhance the production of defense-related proteins to cope the stress. On the other side, we postulated that resistance induced by the chemicals operates via different mechanisms: BABA inducer could act via phenolic biosynthesis pathway, whereas resistance provided by BTH inducer seems to be mediated by defense and stress-related proteins. The results are discussed in terms of response to rust under the effect of inducers.     

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

The influence of wheat (Triticum aestivumL.) resistance, the parasitoid Aphidius rhopalosiphiDe 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.     

Potent inhibition of DNA unwinding and ATPase activities of pea DNA helicase 45 by DNA-binding agents

Pea DNA helicase 45 (PDH45) is an ATP-dependent DNA unwinding enzyme, with intrinsic DNA-dependent ATPase activity [Plant J. 24 (2000) 219]. We have determined the effect of various DNA-binding agents, such as daunorubicin, ethidium bromide, ellipticine, cisplatin, nogalamycin, actinomycin C1, and camptothecin on the DNA unwinding and ATPase activities of the plant nuclear DNA helicase PDH45. The results show that all the agents except actinomycin C1, and camptothecin inhibited the helicase (apparent K(i) values ranging from 1.5 to 7.0 microM) and ATPase (apparent K(i) values ranging from 2.5 to 11.9 microM) activities. This is the first study to show the effect of various DNA-binding agents on the plant nuclear helicase and also first to demonstrate inhibition of any helicase by cisplatin. Another striking finding that the actinomycin C1 and ellipticine act differentially on PDH45 as compared to pea chloroplast helicase suggests that the mechanism of DNA unwinding could be different in nucleus and chloroplast. These results suggest that the intercalation of the inhibitors into duplex DNA generates a complex that impedes translocation of PDH45, resulting in both the inhibitions of unwinding activity and ATP hydrolysis. This study would be useful to obtain a better understanding of the mechanism of plant nuclear DNA helicase unwinding and the mechanism by which these agents can disturb genome integrity.     

Characterization of the metabolite produced by Mycosphaerella pinodes, the causal agent of mycosphaerella blight on field peas (Pisum sativum L.)

The metabolite produced by Mycosphaerella pinodes, the causal agent of mycosphaerella blight on field peas, was detected by thin layer chromatography (TLC) and was analyzed for its chemical and pathogenic characteristics. One blue dot was detected using 254nm UV light on TLC plate, and a spray of rho-anisaldehyde (110 degrees C, 30 min) also produced a blue dot. The solvent systems used for TLC analysis were ethyl acetate/water/acetone (5/2/5), chloroform/methanol/glacial acetic acid (19/10/2), toluene/ethyl acetate/90% formic acid (6/3/1), diethylether/methanol/water/90% formic acid (95/4/1/1), and bezene/methanol/acetic acid (24/2/1), with R(f) values (min-max) of 0.09-0.18, 0.88-0.95, 0.06-0.15, 0.39-0.47 and 0.05-0.12, respectively. The recovered metabolite from the TLC plate displayed UV absorption peaks at 212, 244, 250, 256 and 261 nm. The proposed formula of the main component of the metabolite was C16H12N3O6. The TLC-purified metabolite induced symptom of discoloration on detached pea leaves.     

Inactivation of pea genes by RNAi supports the involvement of two similar O-methyltransferases in the biosynthesis of (+)-pisatin and of chiral intermediates with a configuration opposite that found in (+)-pisatin

(+)-Pisatin, the major phytoalexin of pea (Pisum sativum L.), is believed to be synthesized via two chiral intermediates, (-)-7,2'-dihydroxy-4',5'-methylenedioxyisoflavanone [(-)-sophorol] and (-)-7,2'-dihydroxy-4',5'-methylenedioxyisoflavanol [(-)-DMDI]; both have an opposite C-3 absolute configuration to that found at C-6a in (+)-pisatin. The expression of isoflavone reductase (IFR), which converts 7,2'-dihydroxy-4',5'-methylenedioxyisoflavone (DMD) to (-)-sophorol, sophorol reductase (SOR), which converts (-)-sophorol to (-)-DMDI, and hydroxymaackiain-3-O-methyltransferase (HMM), believed to be the last step of (+)-pisatin biosynthesis, were inactivated by RNA-mediated genetic interference (RNAi) in pea hairy roots. Some hairy root lines containing RNAi constructs of IFR and SOR accumulated DMD or (-)-sophorol, respectively, and were deficient in (+)-pisatin biosynthesis supporting the involvement of chiral intermediates with a configuration opposite to that found in (+)-pisatin in the biosynthesis of (+)-pisatin. Pea proteins also converted (-)-DMDI to an achiral isoflavene suggesting that an isoflavene might be the intermediate through which the configuration is changed to that found in (+)-pisatin. Hairy roots containing RNAi constructs of HMM also were deficient in (+)-pisatin biosynthesis, but did not accumulate (+)-6a-hydroxymaackiain, the proposed precursor to (+)-pisatin. Instead, 2,7,4'-trihydroxyisoflavanone (TIF), daidzein, isoformononetin, and liquiritigenin accumulated. HMM has a high amino acid similarity to hydroxyisoflavanone-4'-O-methyltransferase (HI4'OMT), an enzyme that methylates TIF, an early intermediate in the isoflavonoid pathway. The accumulation of these four compounds is consistent with the blockage of the synthesis of (+)-pisatin at the HI4'OMT catalyzed step resulting in the accumulation of liquiritigenin and TIF and the diversion of the pathway to produce daidzein and isoformononetin, compounds not normally made by pea. Previous results have identified two highly similar "HMMs" in pea. The current results suggest that both of these O-methyltransferases are involved in (+)-pisatin biosynthesis and that one functions early in the pathway as HI4'OMT and the second acts at the terminal step of the pathway.     

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.     

Studies on the nodulation of strain-specific resistant pea lines using single,mixed and delayed inoculation by Rhizobium leguminosarum

Symbiotic interactions between peas and Rhizobium leguminosarum were investigated by inoculating four pea lines, three of which are strain-specific resistant to the European strain 311d, with various combinations of two strains of Rhizobium, 311d and Tom⁺⁺. The strains were almost equally good to infect the susceptible European cultivar Hero when added singly inoculated. After mixed inoculation (1:1 proportion) strain analysis by ELISA revealed that the nodules were preferentially formed by 311d, although some Tom⁺⁺ nodules were also found mainly on the upper part of the root. Our conclusion is that Tom⁺⁺ is less compatible in comparison with 311d. In addition, we found that as the Hero plants emerged, they were becoming more resistant towards infection with not adapted bacteria. The strain-specific resistant lines from Afghanistan belong to two different systems: Afgh. I, completely resistant to 311d and highly nodulating with Tom⁺⁺, and Afgh. III, incompletely resistant to 311d and poorly nodulating with Tom⁺⁺. Mixed inoculations resulted in nodule depressions, as compared to single inoculations with Tom⁺⁺ ranging from 87% to 14%. The ability of 311d to block infection sites on the roots were found to depend on the degree of symbiotic adaptation between Afgh. I and Tom⁺⁺, respectively Afgh. III and Tom⁺⁺. Strain analysis after double strain inoculation of Afgh. I plants revealed that some nodules were induced by strain 311d. Thus, the presence of Tom⁺⁺ in this case influences the degree of host resistance. However, in Afgh. III plants the resistance towards nodulation were unaffected by the presence of Tom⁺⁺. We suggest that the degree of symbiotic adaptation may change the barrier of resistance towards infection.