Nematicidal activity of Ochradenus baccatus against the root‐knot nematode Meloidogyne javanica

Ochradenus baccatus is a widely distributed shrub in desert regions of the Middle East and North Africa. This plant's nematicidal activity against the root‐knot nematode Meloidogyne javanica was evaluated because it has been found to contain exceptionally high levels of glucosinolates. In in vitro assays with aqueous extracts of the plant, 100% of second‐stage juveniles were immobilized after exposure to 4% root‐core extract for 48 h; 8% root‐core extract suppressed their hatching by 87%, whereas stem, flower and root bark showed lower activity. Incorporation of root core or bark into the soil, as fresh or dry powder at 1 and 0·5% (w/w), respectively, reduced the number of nematodes recovered from the soil by 95–100%, whereas the flower and stem were much less effective. Results from further pot experiments indicated that only the root bark consistently contains nematicidal compounds which are effective in soil, whereas the nematicidal activity of the root core in soil was inconsistent. The presence of non‐volatile lipophilic and lipophobic nematicidal compounds in the root bark was suggested by extraction with different polar solvents, but these compounds do not seem to be isothiocyanates – glucosinolate‐hydrolysed compounds with nematicidal activity. Very poor host status of Ochradenus baccatus to M. javanica, M. incognita and M. hapla, but with root‐penetration rates of juveniles similar to those in tomato roots, suggest that this plant may be used as a cover plant or trap plant to reduce nematode populations in the soil.     

The impact of resistant and susceptible wheat cultivars on the multiplication of Heterodera filipjevi and H. avenae in parasite‐infested soil

Cereal cyst nematode (CCN) severely threatens wheat production in many regions of China. Cultivars susceptible to CCN are the main reason for its spread. This study was initiated to determine whether wheat cultivars conferring different resistance levels are the dominant determinants of CCN populations in the rhizospheric soil. Field experiments were conducted at two locations in Henan province, China, where high populations of Heterodera filipjevi or H. avenae have occurred, during the growing seasons of 2010/11 and 2011/12. Conventional enumeration of white female nematodes on the plant roots, reproductive factor (Rf) and a molecular diagnostic approach, PreDicta B test, a soil testing service based on a sensitive quantitative PCR technique, were used to determine the nematode populations in the rhizospheric soils of seven common wheat and durum wheat cultivars with different reactions to CCN. The resistant responses to CCN conferred by durum wheat Wascana and Wakooma and common wheat Madsen were effective against both H. filipjevi and H. avenae and resulted in significantly fewer nematodes (<5 females) on the roots, and lower Rf. Those cultivars were effective in limiting nematode propagation, resulting in fewer CCN eggs in their rhizospheric soils. Taikong 6 conferred moderate resistance (5–10 females) to both Heterodera species. Tianmin 668 only showed resistance to H. avenae. Aikang 58 and Wenmai 19 were susceptible to both CCN species, which facilitated increases in the nematode populations. These results demonstrate that the reactions to CCN of wheat genotypes have obvious impact on the propagation of nematodes.     

Evidence that nematodes may vector the soft rot‐causing enterobacterial phytopathogens

Bacterial soft rot is a globally significant plant disease that causes major losses in the production of many popular crops, such as potato. Little is known about the dispersal and ecology of soft‐rot enterobacteria, and few animals have been identified as vectors for these pathogens. This study investigates whether soil‐living and bacterial‐feeding nematodes could act as vectors for the dispersal of soft‐rot enterobacteria to plants. Soft‐rot enterobacteria associated with nematodes were quantified and visualized through bacterial enumeration, GFP‐tagging, and confocal and electron scanning microscopy. Soft‐rot enterobacteria were able to withstand nematode grazing, colonize the gut of Caenorhabditis elegans and subsequently disperse to plant material while remaining virulent. Two nematode species were also isolated from a rotten potato sample obtained from a potato storage facility in Finland. Furthermore, one of these isolates (Pristionchus sp. FIN‐1) was shown to be able to disperse soft‐rot enterobacteria to plant material. The interaction of nematodes and soft‐rot enterobacteria seems to be more mutualistic rather than pathogenic, but more research is needed to explain how soft‐rot enterobacteria remain viable inside nematodes.     

Long‐lasting β‐aminobutyric acid‐induced resistance protects tomato fruit against Botrytis cinerea

Minimizing losses to pests and diseases is essential for producing sufficient food to feed the world's rapidly growing population. The necrotrophic fungus Botrytis cinerea triggers devastating pre‐ and post‐harvest yield losses in tomato (Solanum lycopersicum). Current control methods are based on the pre‐harvest use of fungicides, which are limited by strict legislation. This investigation tested whether induction of resistance by β‐aminobutyric acid (BABA) at different developmental stages provides an alternative strategy to protect post‐harvest tomato fruit against B. cinerea. Soil‐drenching plants with BABA once fruit had already formed had no impact on tomato susceptibility to B. cinerea. However, BABA application to seedlings significantly reduced post‐harvest infection of fruit. This resistance response was not associated with a yield reduction; however, there was a delay in fruit ripening. Untargeted metabolomics revealed differences between fruit from water‐ and BABA‐treated plants, demonstrating that BABA triggered a defence‐associated metabolomics profile that was long lasting. Targeted analysis of defence hormones suggested a role of abscisic acid (ABA) in the resistance phenotype. Post‐harvest application of ABA to the fruit of water‐treated plants induced susceptibility to B. cinerea. This phenotype was absent from the ABA‐exposed fruit of BABA‐treated plants, suggesting a complex role of ABA in BABA‐induced resistance. A final targeted metabolomic analysis detected trace residues of BABA accumulated in the red fruit. Overall, it was demonstrated that BABA induces post‐harvest resistance in tomato fruit against B. cinerea with no penalties in yield.     

Development of a real‐time PCR method for the detection of the dagger nematodes Xiphinema index,X. diversicaudatum,X. vuittenezi and X. italiae,and for the quantification of X. index numbers

The ectoparasitic dagger nematodes Xiphinema index and Xiphinema diversicaudatum, often at low numbers in the soil, are vectors of grapevine nepoviruses, which cause huge agronomical problems for the vineyard industry. This study reports a method, based on real‐time PCR, for the specific detection of these species and of the closely related non‐vector species Xiphinema vuittenezi and Xiphinema italiae. Specific primers and TaqMan probes were designed from the ribosomal DNA internal transcribed spacer 1 (ITS1), enabling the specific detection of single individuals of each of the X. index, X. diversicaudatum, X. italiae and X. vuittenezi species whatever the nematode population. The specificity of detection and absence of false positive reaction were confirmed in samples of each species mixed with the three other Xiphinema species or mixed with nematodes representative from other genera (non‐plant‐parasitic Dorylaimida, Longidorus sp., Meloidogyne spp., Globodera spp. and Pratylenchus sp.). The method was shown to be valid for the relative quantification of X. index numbers through its use, from crude nematode extracts of soil samples, in a greenhouse assay of grapevine accessions ranging from highly susceptible to resistant. As an alternative to time‐consuming microscopic identification and counting, this real‐time PCR method will provide a fast, sensitive and reliable diagnostic and relative quantification technique for X. index nematodes extracted from fields or controlled conditions.     

Inoculation of Malus genotypes with a set of Erwinia amylovora strains indicates a gene‐for‐gene relationship between the effector gene eop1 and both Malus floribunda 821 and Malus ‘Evereste’

The Gram‐negative bacterium Erwinia amylovora, causal agent of fire blight disease in pome fruit trees, encodes a type three secretion system (T3SS) that translocates effector proteins into plant cells that collectively function to suppress host defences and enable pathogenesis. Until now, there has only been limited knowledge about the interaction of effector proteins and host resistance presented in several wild Malus species. This study tested disease responses in several Malus wild species with a set of effector deletion mutant strains and several highly virulent E. amylovora strains, which are assumed to influence the host resistance response of fire blight‐resistant Malus species. The findings confirm earlier studies that deletion of the T3SS abolished virulence of the pathogen. Furthermore, a new gene‐for‐gene relationship was established between the effector protein Eop1 and the fire blight resistant ornamental apple cultivar Evereste and the wild species Malus floribunda 821. The results presented here provide new insights into the host–pathogen interactions between Malus sp. and E. amylovora.     

Genetic diversity of the root‐knot nematode Meloidogyne ethiopica and development of a species‐specific SCAR marker for its diagnosis

Meloidogyne ethiopica is an important nematode pathogen causing serious economic damage to grapevine in Chile. In Brazil, M. ethiopica has been detected with low frequency in kiwifruit and other crops. The objectives of this study were to evaluate the intraspecific genetic variability of M. ethiopica isolates from Brazil and Chile using AFLP and RAPD markers and to develop a species‐specific SCAR‐PCR assay for its diagnosis. Fourteen isolates were obtained from different geographic regions or host plants. Three isolates of an undescribed Meloidogyne species and one isolate of M. ethiopica from Kenya were included in the analysis. The results showed a low level of diversity among the M. ethiopica isolates, regardless of their geographical distribution or host plant origin. The three isolates of Meloidogyne sp. showed a high homogeneity and clustered separately from M. ethiopica (100% bootstrap). RAPD screenings of M. ethiopica allowed the identification of a differential DNA fragment that was converted into a SCAR marker. Using genomic DNA from pooled nematodes as a template, PCR amplification with primers designed from this species‐specific SCAR produced a fragment of 350 bp in all 14 isolates of M. ethiopica tested, in contrast with other species tested. This primer pair also allowed successful amplification of DNA from single nematodes, either juveniles or females and when used in multiplex PCR reactions containing mixtures of other root‐knot nematode species, thus showing the sensitivity of the assay. Therefore, the method developed here has potential for application in routine diagnostic procedures.     

重要植物寄生线虫内切葡聚糖酶基因研究进展

 在线虫与植物互作的过程中,降解寄主细胞壁是植物线虫成功建立寄生关系的关键环节。β-1,4-内切葡聚糖酶(β-1,4-endoglucanase,ENG)是由线虫食道腺细胞产生并由口针分泌、对细胞壁降解起关键作用的酶类之一。本文对近年来植物寄生线虫eng基因的克隆、组织定位、表达分析、基因、编码蛋白的结构功能以及ENG来源、进化及在线虫与植物互作中的潜在作用等进行了概述。     

Effects of pyrrolizidine alkaloids on the performance of plant‐parasitic and free‐living nematodes

BACKGROUND: Chemical nematicides such as methyl bromide have for decades played a significant role in the management of plant‐parasitic nematodes. Their application is problematic because of negative environmental impacts, and therefore methyl bromide was phased out in Europe in 2005. A possible alternative to synthetically derived nematicides is seen in the use of plants and/or their secondary metabolites. These plants could either be used as nematicidal green manure or as a source for nematicidal extracts. This study aimed to evaluate the effects of 1,2‐dehydropyrrolizidine alkaloids (PAs), a group of secondary plant metabolites found in hundreds of plant species throughout the world, on the performance of plant‐parasitic and free‐living nematodes. RESULTS: PAs induced nematicidal, ovicidal and repellent effects on different plant‐parasitic and free‐living nematodes. There was no conclusive ranking in toxicity for the different structural types of PAs tested. However, the effects were often more pronounced for the tertiary than for the oxidised form of PAs. Further, large differences were observed in the susceptibility of different nematode species to PAs. CONCLUSIONS: PAs do affect several performance parameters and developmental stages of nematodes. Therefore, PA‐producing plants such as species of Crotalaria, Ageratum or Senecio might be promising candidates for nematode management strategies. [Correction made here after initial online publication] Copyright © 2009 Society of Chemical Industry     

The β‐amylase genes: negative regulators of disease resistance for Verticillium dahliae

Little is known about the role of plant primary metabolism in defence against pathogens. The present study is the first investigation published that examines the role of β‐amylase (BAM) genes upon fungal, Verticillium dahliae, infection. The responses of Arabidopsis thaliana plants impaired in BAM1, BAM2, BAM3, BAM4 genes, along with double, triple and quadruple mutants of those genes, were used to explore the involvement of BAM in the host plant–V. dahliae interaction. Less severe symptoms were recorded in bam mutants compared to wild type. Real‐time quantitative PCR (qPCR) revealed that the decrease in symptom severity shown in bam plants was correlated with reductions in the growth of the pathogen in the plants. Confocal microscopy of the most and least susceptible bam mutants and the wildtype plants showed that there were no differences between them in the number of attached conidia and penetration sites on the roots. BAM1, BAM2 and BAM3 expression was altered upon V. dahliae infection in the aerial tissues of the wild type. Analysis by qPCR of the PR1 and PDF1.2 expression in the bam3, bam1234, bam14 and wildtype plants showed that PR1 was up‐regulated in the roots of bam plants upon V. dahliae infection.     

Selection of nematodes by resistant plants has implications for local adaptation and cross‐virulence

The variability of resistance durability in different potato genotypes harbouring the same resistance QTL but differing by their genetic background was explored. The indirect consequences of the resistance adaptation in terms of local (i.e. genotype‐specific) adaptation and cross‐virulence was also investigated. Following the virulence of the potato cyst nematode Globodera pallida in a long‐term experimental evolution protocol, the results showed that nematode populations were able to adapt to the resistance of four potato genotypes carrying the QTL GpaV from Solanum vernei, and that the plant genetic background has an impact upon the durability of resistance. The pattern of local adaptation observed here indicates that divergent selection has occurred during the experimental evolution performed from the same initial nematode population, and revealed a trade‐off between the adaptation to a resistant potato genotype and the adaptation to another resistant genotype differing in its genetic background. In terms of cross‐virulence between potato genotypes derived from different resistance sources (S. sparsipilum and S. spegazzinii), this study shows that the adaptation to resistance QTL GpaV_vrn does not necessarily allow the adaptation to collinear GpaV loci. The results presented here could be useful for predicting evolution of nematode populations in natural agro‐ecosystems and identifying durable strategies for resistance deployment.     

Rapid detection and quantification of viable potato cyst nematodes using qPCR in combination with propidium monoazide

Potato cyst nematodes (PCN), Globodera pallida and Globodera rostochiensis, are obligate parasites of solanaceous plants, causing severe losses in several potato growing areas throughout the world. To date, management of PCN is related to nematode population densities estimated as eggs per gram of soil, without considering the actual number of viable juveniles within the cysts. In classical nematology, the standard method to determine PCN viability is based on a staining assay, using Meldola's blue dye (MB) followed by microscopic visualization of MB‐treated nematodes. Although MB is considered to be reliable in staining embryonated juveniles within eggs and cysts, it is a time‐ and labour‐consuming assay. In the present work, a real‐time PCR (qPCR)‐based method combined with propidium monoazide (PMA), a photoreactive DNA‐intercalating dye, was developed for the quantification of viable PCN. This dye renders exposed DNA of dead cells unable to be amplified by PCR, and thus only DNA from viable/intact PCN juveniles is amplified and detected. The novelty of the present method lies in the simultaneous quantitative and qualitative estimation of viable PCN inocula using species‐specific primers and TaqMan probes. The PMA–qPCR viability method (v‐PCR) developed for the two Globodera species successfully discriminated dead from living specimens in heat‐treated samples and eggs in old and newly formed cysts. Interestingly, the detection of DNA from 34‐year‐old nematode cysts stored at room temperature was observed. In conclusion, the proposed v‐PCR method should prove to be very useful for the routine determination of PCN viability from field samples.     

Characterization and functional analysis of clpB gene from Acidovorax avenae subsp. avenae RS‐1

The type VI secretion system (T6SS) has been reported to be highly associated with various cellular activities in strain RS‐1 of Acidovorax avenae subsp. avenae (Aaa), the pathogen of bacterial brown stripe of rice. However, the role of the clpB gene that presents in the T6SS gene cluster in Aaa pathogenicity has not been clarified. The aim of the current study was to characterize the function of clpB and to investigate its contribution to bacterial pathogenesis using insertional deletion mutation and complementation approaches. The results indicated that mutation of clpB significantly affected bacterial growth, virulence, exopolysaccharide (EPS) production, biofilm formation and expression of 13 other T6SS genes of Aaa RS‐1. The reduction of virulence may be also partially due to the change in EPS composition, which was characterized by the Fourier transform infrared (FTIR) spectra. Furthermore, analysis of protein homology modelling showed that the structure of ClpB is different from those of the other T6SS components. In addition, structural difference was observed between ClpB and Type IV pili (TFP) as well as Type IV pilus biogenesis proteins (PilP), whose functions are similar to ClpB. Taken together, this study demonstrated that the clpB gene plays a key role in Aaa bacterial virulence.     

Prevalence and diversity of plant parasitic nematodes in Northern Ireland grassland and cereals,and the influence of soils and rainfall

The prevalence and diversity of plant parasitic nematodes in Northern Ireland cereal and grassland was determined from 191 agricultural fields. A total of 18 nematode genera were detected, including economically important pests, Meloidogyne spp., Heterodera spp. and Pratylenchus spp., each of which were above economic damage thresholds in a significant proportion of the sites (92.4%, 70% and 28.6%, respectively). The detection of the root knot nematode, Meloidogyne minor (6% prevalence), was significant given its recent emergence across the turf grass sector and the prospect of M. minor becoming a common agricultural pest. Analyses of nematode prevalence and abundance highlighted significant associations with grass and cereals, soil types, soil grade (proxy for soil quality) and rainfall levels. Specifically, nematode populations varied between the two major soils (brown earths and gleys), while significant trends for increased nematode diversity and greater prevalence of both Meloidogyne and Pratylenchus with increasing rainfall were also observed. Multivariate analyses were performed to determine interactive effects and the relative importance of the factors affecting nematode populations. Notably, rainfall, in combination with either crop type or soil grade, had a significant effect on nematode abundance and diversity. The findings suggest significant changes in nematode populations have occurred over the last several decades and the possibility that these are linked to changing climate and cropping practices are discussed, as well as future concerns for plant parasitic nematode management.     

Differential metabolic response of barley genotypes,varying in resistance,to trichothecene‐producing and ‐nonproducing (tri5−) isolates of Fusarium graminearum

Resistance of barley to Fusarium graminearum was studied using a pair each of resistant and susceptible black and yellow barley lines. The spikelets were inoculated with a trichothecene‐producing isolate, a trichothecene‐nonproducing isolate (tri5^?), or a mock solution. Spikelets were collected 72 h after inoculation and metabolites were analysed using a LC‐hybrid MS system. Metabolite abundances were used to identify the constitutive (RRC) and induced resistance‐related metabolites (RRI). The pathogen virulence factor, DON, and its plant detoxification product, DON‐3‐O‐glucoside (D3G), were also identified and designated as resistance‐indicator (RI) metabolites. The RRC, RRI and RI metabolites were putatively identified. Jasmonic acid was significantly induced in barley following inoculation with a trichothecene‐producing isolate, but not with a tri5^? isolate. The former isolate reduced the induction of both the number and amount of RR metabolites. The metabolites cinnamic acid, sinapoyl alcohol, coniferin, catechin and naringin were identified only in response to the inoculation with a tri5^? mutant. The abundances of p‐coumaric acid, coniferaldehyde and sinapaldehyde increased more in response to the tri5^? mutant than to the trichothecene‐producing isolate. The total amount of DON synthesized and its conversion to D3G varied greatly between the resistant and susceptible black barley, but not in yellow barley. Interestingly, an increase in the amount of total DON produced was associated with a decrease in the conversion of DON to D3G. The roles of RRC, RRI and RI metabolites in plant defence and their further use as potential biomarkers in screening are discussed.