Genotypic and phenotypic diversity of a baculovirus population within an individual insect host

It is becoming increasingly apparent that many pathogen populations, including those of insects, show high levels of genotypic variation. Baculoviruses are known to be highly variable, with isolates collected from the same species in different geographical locations frequently showing genetic variation and differences in their biology. More recent Studies at smaller scales have also shown that virus DNA profiles from individual larvae can show polymorphisms within and between populations of the same species. Here, we investigate the genotypic and phenotypic variation of an insect baculovirus infection within a single insect host. Twenty four genotypically distinct nucleopolyhedrovirus (NPV) variants were isolated from an individual pine beauty moth, Panolis flammea, caterpillar by in vivo cloning techniques. No variant appeared to be dominant in the population. The Pafl NPV variants have been mapped using three restriction endonucleases and shown to contain three hypervariable regions containing insertions of 70-750 bp. Comparison of seven of these variants in an alternative host, Mamestra brassicae, demonstrated that the variants differed significantly in both pathogenicity and speed of kill. The generation and maintenance of pathogen heterogeneity are discussed. (c) 2005 Elsevier Inc. All rights reserved.

Interazioni piante - insetti: ruolo delle formiche in un sistema multitrofico. Aspetti ecoetologici ed applicativi

Le formiche svolgono un importante ruolo all’interno degli ecosistemi ed alcune specie sono considerate keystone in quanto in grado di modificare la componente biotica e/o abiotica dell’ecosistema stesso. Sono animali ubiquitari che hanno colonizzato molteplici ambienti, compresi gli agroecosistemi. Negli agroecosistemi spesso svolgono un ruolo impattante determinando la diffusione o il regresso di specie di artropodi, alcune delle quali dannose alle colture. La presente ricerca tiene conto di un’ampia visione dei rapporti ecoetologici intercorrenti tra le formiche e la componente biotica di un ecosistema, utilizzando il concetto di rete multitrofica. In quest’ottica, si è pensato di costruire un sistema multitrofico costituito da una specie vegetale di interesse agrario (Cucumis sativus), dai suoi fitofagi naturali, divisi in fitomizi (afidi) (Aphis gossypii e Myzus persicae) e fitofagi masticatori (bruchi del lepidottero Mamestra brassicae), formiche (Formica pratensis) e predatori afidofagi (Aphidolets aphidimyza). Il sistema multitrofico è stato utilizzato sia per studiare l’aggressività delle formiche, sia per verificare l’esistenza di una comunicazione interspecifica tra le formiche e le piante (allelochimici). Gli studi sull’aggressività sono consistiti nel: • Verificare il livello di aggressività delle formiche nei confronti di un fitofago masticatore, competitore degli afidi nello sfruttare la pianta ospite. • Verificare se la presenza di afidi mutualisti fa variare il livello di aggressività delle formiche verso il competitore. • Verificare se esiste aggressività verso un predatore di afidi, i quali, secondo il paradigma della trofobiosi, dovrebbero essere difesi dalle formiche in cambio della melata. • Verificare se il predatore ha evoluto strategie volte ad eludere il controllo delle formiche sugli insetti che si approcciano alla colonia di afidi. Gli studi sui rapporti piante-formiche sono stati effettuati mediante olfattometro, osservando la risposta delle formiche alle sostanze volatili provenienti da piante infestate in modo differente con i fitofagi del sistema. Attraverso il trappolaggio e l’analisi gas-cromatografica delle sostanze prodotte dalle piante oggetto di studio abbiamo quindi individuato tipo e quantità di ogni composto volatile. Oltre alle piante di cetriolo, per questi esperimenti sono state utilizzate anche piante di patata (Solanum tuberosum). Dagli esperimenti sull’aggressività è risultato che le formiche manifestano un elevato potenziale predatorio, eradicando completamente la presenza dei bruchi sulle piante. Questo livello di aggressività tuttavia non cresce con la presenza degli afidi mutualisti che dovrebbero essere difesi dai competitori. Le formiche inoltre non sono in grado di sopprimere i predatori afidofagi che ipotizziamo riescano ad effettuare un camuffamento chimico, assumendo gli odori degli afidi dei quali si nutrono. I risultati degli esperimenti in olfattometro mostrano una chiara risposta positiva delle formiche verso gli odori di alcune delle piante infestate. Vi sono delle differenze nella risposta in funzione della specie di fitofago presente e della specie di pianta utilizzata. Nei trattamenti in cui erano presenti le piante di C. sativus, gli esperimenti in olfattometro hanno mostrato che le formiche rispondono in modo significativo agli odori emessi dalle piante in cui vi era la presenza del fitofago masticatore M. brassicae, solo o in associazione con A. gossypii. La presenza dei soli afidi, sia mutualisti (A. gossypii) sia non mutualisti (M. persicae), non ha invece indotto una risposta significativa nelle formiche rispetto agli odori delle piante non infestate. Nei trattamenti in cui erano presenti le piante di S. tuberosum la scelta delle formiche è stata significativa verso gli odori emessi dalle piante infestate con ciascuna delle singole specie di erbivori rispetto alle piante non infestate. Gli esperimenti sull’analisi delle sostanze volatili emesse dalle piante hanno confermato che gli organismi vegetali sono una vera centrale di produzione biochimica, infatti ben 91 composti volatili diversi sono stati individuati dall’analisi gas-cromatografica delle piante di cetriolo e 85 in quelle di patata. Dalle elaborazioni effettuate, rispettivamente 27 e 4 di essi sono prodotti esclusivamente dalle piante attaccate dai fitofagi. In generale, il cambiamento più consistente è dato dalla quantità di alcune sostanze volatili emesse dalle piante infestate rispetto a quelle integre che determina un cambiamento nei rapporti tra le sostanze che compongono i volatiles. E’ probabile che l’effetto attrattivo esercitato sulle formiche sia dato da un Blend di sostanze più che dai singoli composti presenti

Oviposition and reproductive performance of a generalist parasitoid (Trichogramma pretiosum) exposed to host species that differ in their physical characteristics

The response of generalist egg parasitoids to alternative natural hosts that are present simultaneously is not well known. We investigated the behavior of Trichogramma pretiosum Riley (Hymenoptera: Trichogrammatidae) in relation to two field hosts Helicoverpa armigera Hubner and Spodoptera litura Fabricius, in choice and no choice tests. We quantified the effects of natal host species and post-emergence adult age on the oviposition preference of the parasitoids. H. armigera eggs were consistently preferred over S. litura eggs, regardless of the natal host and adult age. When only S. litura eggs were available as hosts, they were parasitized at statistically similar rates to H. armigera eggs (average of 17 +/- 2.7 vs. 13 +/- 3.0, H. armigera to S. litura). The adult lifespan and lifetime fecundity of T. pretiosum were variable but were affected by natal host species and/or host species to which they were exposed. Mean lifespan and fecundity of parasitoids that had developed in H. armigera eggs and were exposed to H. armigera eggs for oviposition were 13.9 +/- 1.8 days and 98.7 +/- 11.0 adult offspring. By contrast, those that developed in S. litura eggs and were exposed to S. litura eggs for oviposition lived for 7 +/- 0.9 days and produced 53.8 +/- 8.0 adult offspring. The ovigeny index (OI) was significantly lower in the parasitoids exposed to H. armigera eggs than in those exposed to S. litura eggs, regardless of the natal host, indicating that H. armigera eggs sustain the adult parasitoids better than S. litura eggs. These results are used to predict parasitoid behavior in the field when both hosts are available. (c) 2006 Elsevier Inc. All rights reserved.

Efecto de la temperatura sobre el pulgón del repollo (Brevicoryne brassicae L.) (Homóptera : Aphidoidea)

p.99-103

Biología y demografía de Brevicoryne brassicae (L.) (Homoptera: Aphididae) sobre cuatro cultivares de Brassica oleracea L.

p.87-91

Effect of Bacillus thuringiensis naturally colonising Brassica campestris var. chinensis leaves on neonate larvae of Pieris brassicae

The feeding of neonate larvae of Pieris brassicae (Order Lepidoptera) on leaves of brassica plants that had been colonised by Bacillus thuringiensis resulted in the death of 35% of the population within 72 h. The bacteria Multiplied in the cadavers, resulting in an increase of about 50-fold compared to the living insects. Surviving insects showed no ill effects during the time of the study. There was negligible multiplication of B. thuringiensis in the frass. (C) 2009 Elsevier Inc. All rights reserved.

The ecology of Bacillus thuringiensis on the Phylloplane: colonization from soil, plasmid transfer, and interaction with larvae of Pieris brassicae

Seedlings of clover (Triflorium hybridum) were colonized by Bacillus thuringiensis when spores and seeds were co-inoculated into soil. Both a strain isolated in the vegetative form from the phylloplane of clover, 2810-S-4, and a laboratory strain, HD-1, were able to colonize clover to a density of about 1000 CFU/g leaf when seeds were sown in sterile soil and to a density of about 300 CFU/g leaf in nonsterile soil. A strain lacking the characteristic insecticidal crystal proteins produced a similar level of colonization over a 5-week period as the wild type strain, indicating that crystal production was not a mitigating factor during colonization. A small plasmid, pBC16, was transferred between strains of B. thuringiensis when donor and recipient strains were sprayed in vegetative form onto leaves of clover and pak choi (Brassica campestris var. chinensis). The rate of transfer was about 0.1 transconjugants/recipient and was dependent on the plant species. The levels of B. thuringiensis that naturally colonized leaves of pak choi produced negligible levels of mortality in third instar larvae of Pieris brassicae feeding on the plants. Considerable multiplication occurred in the excreted frass but not in the guts of living insects. Spores in the frass could be a source of recolonization from the soil and be transferred to other plants. These findings illustrate a possible cycle, not dependent on insect pathology, by which B. thuringiensis diversifies and maintains itself in nature.

The occurrence and economic impact of plasmodiophora brassicae and clubroot disease

The significance of Plasmodiophora brassicae Woronin and clubroot disease which it incites in members of the family Brassicaceae is reviewed as the focus for this special edition of the Journal of Plant Growth Regulation. This is a monographic treatment of recent research into the pathogen and disease; previous similar treatments are now well over half a century old. Vernacular nomenclature of the disease indicates that it had a well-established importance in agriculture and horticulture from at least the Middle Ages onward in Europe and probably earlier. Subsequently, the pathogen probably spread worldwide as a result of transfer on and in fodder taken by colonists as livestock feed. It is a moot point, however, whether there was much earlier spread by P. brassicae into China and subsequently Japan as Brassica rapa (Chinese cabbage and many variants) colonized those lands in archaeological time. Symptoms, worldwide distribution, and economic impact are briefly described here to provide a basis for understanding subsequent papers. Clubroot disease devastates both infected field and protected vegetable and agricultural Brassica crops. Particular importance is placed on recent reports of crop losses in tropical countries, albeit where the crops are grown in cooler altitudes, and in the Canadian prairie land canola crops. The latter is of enormous importance because this crop is the single most important and essential source of vegetable oils used in human foodstuffs and in industrial lubricants where mineral oils are inappropriate.

Plasmodiophora brassicae in its Environment

Plasmodiophora brassicae Wor. is viewed in this article from the standpoint of a highly evolved and successful organism, well fitted for the ecological niche that it occupies. Physical, chemical, and biological components of the soil environment are discussed in relation to their effects on the survival, growth, and reproduction of this microbe. It is evident that P. brassicae is well equipped by virtue of its robust resting spores for survival through many seasonal cycles. Germination is probably triggered as a result of signals initiated by root exudates. The resultant motile zoospore moves rapidly to the root hair surface and penetration and colonization follow. The short period between germination and penetration is one of greatest vulnerability for P. brassicae. In this phase survival is affected at the very least by soil texture and structure; its moisture; pH; calcium, boron, and nitrogen content; and the presence of active microbial antagonists. These factors influence the inoculum potential (sensu Garrett, 1956) and its viability and invasive capacity. There is evidence that these effects may also influence differentially the survival of some physiologic races of P. brassicae. Considering the interaction of P. brassicae with the soil environment from the perspective of its biological fitness is an unusual approach; most authors consider only the opportunities to destroy this organism. The approach adopted here is borne of several decades spent studying P. brassicae and the respect that has been engendered for it as a biological entity. This review stops at the point of penetration, although some of the implications of the environment for successful colonization are included because they form a continuum. Interactions with the molecular and biochemical cellular environment are considered in other sections in this special edition.

Detection and molecular characterisation of Pyrenopeziza brassicae isolates resistant to methyl benzimidazole carbamates

BACKGROUND Methyl benzimidazole carbamate (MBC) fungicides are used to control the oilseed rape pathogen Pyrenopeziza brassicae. Resistance to MBCs has been reported in P. brassicae, but the molecular mechanism(s) associated with reductions in sensitivity have not been verified in this species. Elucidation of the genetic changes responsible for resistance, hypothesised to be target-site mutations in β-tubulin, will enable resistance diagnostics and thereby inform resistance management strategies. RESULTS P. brassicae isolates were classified as sensitive, moderately resistant or resistant to MBCs. Crossing P. brassicae isolates of different MBC sensitivities indicated that resistance was conferred by a single gene. The MBC-target encoding gene β-tubulin was cloned and sequenced. Reduced MBC sensitivity of field isolates correlated with β-tubulin amino acid substitutions L240F and E198A. The highest level of MBC resistance was measured for isolates carrying E198A. Negative cross-resistance between MBCs and the fungicides diethofencarb and zoxamide was only measured in E198A isolates. PCR-RFLP was used to screen isolates for the presence of L240F and E198A. The substitutions E198G and F200Y were also detected in DNA samples from P. brassicae populations after cloning and sequencing of PCR products. The frequencies of L240F and E198A in different P. brassicae populations were quantified by pyrosequencing. There were no differences in the frequencies of these alleles between P. brassicae populations sampled from different locations or after fungicide treatment regimes. CONCLUSIONS The molecular mechanisms affecting sensitivity to MBCs in P. brassicae have been identified. Pyrosequencing assays are a powerful tool for quantifying fungicide-resistant alleles in pathogen populations.

Alterations in predicted regulatory and coding regions of the sterol 14α-demethylase gene (CYP51) confer decreased azole sensitivity in the oilseed rape pathogen Pyrenopeziza brassicae

The incidence and severity of light leaf spot epidemics caused by the ascomycete fungus Pyrenopeziza brassicae on UK oilseed rape crops is increasing. The disease is currently controlled by a combination of host resistance, cultural practices and fungicide applications. We report decreases in sensitivities of modern UK P. brassicae isolates to the azole (imidazole and triazole) class of fungicides. By cloning and sequencing the P. brassicae CYP51 (PbCYP51) gene, encoding the azole target sterol 14α-demethylase, we identified two non-synonymous mutations encoding substitutions G460S and S508T associated with reduced azole sensitivity. We confirmed the impact of the encoded PbCYP51 changes on azole sensitivity and protein activity by heterologous expression in a Saccharomyces cerevisiae mutant YUG37::erg11 carrying a controllable promoter of native CYP51 expression. In addition, we identified insertions in the predicted regulatory regions of PbCYP51 in isolates with reduced azole sensitivity. The presence of these insertions was associated with enhanced transcription of PbCYP51 in response to sub-inhibitory concentrations of the azole fungicide tebuconazole. Genetic analysis of in vitro crosses of sensitive and resistant isolates confirmed the impact of PbCYP51 alterations in coding and regulatory sequences on a reduced sensitivity phenotype, as well as identifying a second major gene at another locus contributing to resistance in some isolates. The least sensitive field isolates carry combinations of upstream insertions and non-synonymous mutations, suggesting PbCYP51 evolution is on-going and the progressive decline in azole sensitivity of UK P. brassicae populations will continue. The implications for the future control of light leaf spot are discussed.

Premature germination of resting spores as a means of Protecting brassica crops from Plasmodiophora brassicae Wor., (clubroot)

Premature germination of resting spores as a means of protecting brassica crops from Plasmodiphora brassicae Wor., (Clubroot). Crop Protection. Clubroot disease causes substantial yield and quality losses in broadacre oil seed and intensive vegetable brassica crops worldwide. The causal microbe Plasmodiophora brassicae Wor., perennates as soil-borne dormant resting spores. Their germination is triggered by exudates from host roots. A valuable addition to sustainable integrated control strategies could be developed by identifying and synthesising the molecules responsible for stimulating resting spore germination. This paper reports experiments in which stimulatory exudates were collected from brassica roots following exposure to infective stages of P. brassicae. Analyses identified a germination signalling molecule of circa 1 kDa formed of glucose sub-units. Mass spectral analyses showed this to be a complex hexasaccharide carbohydrate with structural similarities to the components of plant cell walls. This is the first report of a host generated hexasaccharide which is capable of stimulating the germination of resting spores of P. brassicae. The implications for environmentally benign control of clubroot are discussed briefly.

Clubroot (Plasmodiophora brassicae Woronin): an agricultural and biological challenge worldwide

Clubroot disease and the causal microbe Plasmodiophora brassicae offer abundant challenges to agriculturists and biological scientists. This microbe is well fitted for the environments which it inhabits. Plasmodiophora brassicae exists in soil as microscopic well protected resting spores and then grows actively and reproduces while shielded inside the roots of host plants. The pathogen is active outside the host for only short periods. Consequently, scientific studies are made challenging by the biological context of the host and pathogen and the technology required to investigate and understand that relationship. Controlling clubroot disease is a challenge for farmers, crop consultants and plant pathology practitioners because of the limited options which are available. Full symptom expression happens solely in members of the Brassicaceae family. Currently, only a few genes expressing strong resistance to P. brassicae are known and readily available. Agrochemical control is similarly limited by difficulties in molecule formulation which combines efficacy with environmental acceptability. Manipulation of husbandry encouraging improvements in soil structure, texture, nutrient composition and moisture content can reduce populations of P. brassicae. Integrating such strategies with rotation and crop management will reduce but not eliminate this disease. There are indications that forms of biological competition may be mobilised as additions to integrated control strategies. The aim of this review is to chart key themes in the development of scientific biological understanding of this host-pathogen relationship by offering signposts to grapple with clubroot disease which devastates crops and their profitability. Particular attention is given to the link between soil and nutrient chemistry and activity of this microbe.