Fusarium wilt of lettuce, caused worldwide by Fusarium oxysporum f. sp. lactucae, is an emerging seed-transmitted disease on Lactuca sativa. In order to develop a molecular diagnostic tool for identifying race 1 (VCG0300) of the pathogen on vegetable samples, an effective technique is presented. Inter-retrotransposon amplified polymorphism polymerase chain reaction (PCR), a technique based on the amplification of genomic regions between long terminal repeats, was applied. It was shown to be useful for grouping F. oxysporum f. sp. lactucae race 1 isolates. Inter-retrotransposon sequence-characterized amplified regions (IR-SCAR) was used to develop a specific set of PCR primers to be utilized for differentiating F. oxysporum f. sp. lactucae isolates from other F. oxysporum isolates. The specific primers were able to uniquely amplify fungal genomic DNA from race 1 isolates obtained in Italy, Portugal, the United States, Japan, and Taiwan. The primers also were specific to pathogen DNA obtained from artificially infected lettuce seed and naturally and artificially infected plants.

BackgroundLarvae of the Western Corn Rootworm (WCR) feeding on maize roots cause heavy economical losses in the US and in Europe. New or adapted pest management strategies urgently require a better understanding of the multitrophic interaction in the rhizosphere. This study aimed to investigate the effect of WCR root feeding on the microbial communities colonizing the maize rhizosphere.Methodology/Principal FindingsIn a greenhouse experiment, maize lines KWS13, KWS14, KWS15 and MON88017 were grown in three different soil types in presence and in absence of WCR larvae. Bacterial and fungal community structures were analyzed by denaturing gradient gel electrophoresis (DGGE) of the16S rRNA gene and ITS fragments, PCR amplified from the total rhizosphere community DNA. DGGE bands with increased intensity were excised from the gel, cloned and sequenced in order to identify specific bacteria responding to WCR larval feeding. DGGE fingerprints showed that the soil type and the maize line influenced the fungal and bacterial communities inhabiting the maize rhizosphere. WCR larval feeding affected the rhiyosphere microbial populations in a soil type and maize line dependent manner. DGGE band sequencing revealed an increased abundance of Acinetobacter calcoaceticus in the rhizosphere of several maize lines in all soil types upon WCR larval feeding.Conclusion/SignificanceThe effects of both rhizosphere and WCR larval feeding seemed to be stronger on bacterial communities than on fungi. Bacterial and fungal community shifts in response to larval feeding were most likely due to changes of root exudation patterns. The increased abundance of A. calcoaceticus suggested that phenolic compounds were released upon WCR wounding.

BackgroundThe western corn rootworm (WCR) is one of the economically most important pests of maize. A better understanding of microbial communities associated with guts and eggs of the WCR is required in order to develop new pest control strategies, and to assess the potential role of the WCR in the dissemination of microorganisms, e.g., mycotoxin-producing fungi.Methodology/Principal FindingsTotal community (TC) DNA was extracted from maize rhizosphere, WCR eggs, and guts of larvae feeding on maize roots grown in three different soil types. Denaturing gradient gel electrophoresis (DGGE) and sequencing of 16S rRNA gene and ITS fragments, PCR-amplified from TC DNA, were used to investigate the fungal and bacterial communities, respectively. Microorganisms in the WCR gut were not influenced by the soil type. Dominant fungal populations in the gut were affiliated to Fusarium spp., while Wolbachia was the most abundant bacterial genus. Identical ribosomal sequences from gut and egg samples confirmed a transovarial transmission of Wolbachia sp. Betaproteobacterial DGGE indicated a stable association of Herbaspirillum sp. with the WCR gut. Dominant egg-associated microorganisms were the bacterium Wolbachia sp. and the fungus Mortierella gamsii. Conclusion/SignificanceThe soil type-independent composition of the microbial communities in the WCR gut and the dominance of only a few microbial populations suggested either a highly selective environment in the gut lumen or a high abundance of intracellular microorganisms in the gut epithelium. The dominance of Fusarium species in the guts indicated WCR larvae as vectors of mycotoxin-producing fungi. The stable association of Herbaspirillum sp. with WCR gut systems and the absence of corresponding sequences in WCR eggs suggested that this bacterium was postnatally acquired from the environment. The present study provided new insights into the microbial communities associated with larval guts and eggs of the WCR. However, their biological role remains to be explored.

Fusarium oxysporum f. sp. lactucae, the causal agent of Fusarium wilt of lettuce, has been reported in three continents in the last 10 years. Forty-seven isolates obtained from infected plants and seed in Italy, the United States, Japan, and Taiwan were evaluated for pathogenicity and vegetative compatibility. Chlorate-resistant, nitrate-nonutilizing mutants were used to determine genetic relatedness among isolates from different locations. Using the vegetative compatibility group (VCG) approach, all Italian and American isolates, type 2 Taiwanese isolates, and a Japanese race 1 were assigned to the major VCG 0300. Taiwanese isolates type 1 were assigned to VCG 0301. The hypothesis that propagules of Fusarium oxysporum f. sp. lactucae that caused epidemics on lettuce in 2001-02 in Italian fields might have spread via import and use of contaminated seeds is discussed.

Lysyl-phosphatidylglycerol is one of the components of the mycobacterial membrane that contributes to the resistance to cationic antimicrobial peptides, a host-induced frontline defense against invading pathogens. Its production is catalyzed by LysX, a bifunctional protein with lysyl transferase and lysyl transfer RNA synthetase activity. Comparative proteome analysis of a lysX mutant of Mycobacterium avium strain 104 and the wild type indicated that the lysX mutant strain undergoes a transition in phenotype by switching the carbon metabolism to β-oxidation of fatty acids, along with accumulation of lipid inclusions. Surprisingly, proteins associated with intracellular survival were upregulated in the lysX mutant, even during extracellular growth, preparing bacteria for the conditions occurring inside host cells. In line with this, the lysX mutant exhibited enhanced intracellular growth in human-blood-derived monocytes. Thus, our study exposes the significance of lysX in the metabolism and virulence of the environmental pathogen M. avium hominissuis.