Widespread distribution of Xanthomonas perforans and limited presence of X. gardneri in Brazil

Tomato bacterial spot is caused by Xanthomonas euvesicatoria, X. vesicatoria, X. perforans and X. gardneri. In order to determine the distribution, frequency of occurrence, and diversity of these species in the Brazilian commercial tomato fields, a survey was conducted between 2009 and 2012. In this period, 204 strains were obtained from 33 counties (22 with processing tomatoes and 11 with fresh‐market tomatoes). Pathogenicity tests, BOX‐PCR, PCR with species‐specific primers, and sequence analysis of the avirulence gene avrXv3 were performed in order to identify the strains at species and race level. Xanthomonas perforans predominated among the strains (92%) and was present in most counties. In addition, this species was prevalent in most areas of both fresh‐market tomatoes (63.6% of counties surveyed) and processing tomatoes (95.4% of counties surveyed). Fifteen strains (7.5%) were identified as X. gardneri, which was found mostly in fresh‐market fields located at regions with altitude higher than 900 m, and only one strain of X. euvesicatoria (0.5%) was found in a processing tomato field. High genetic diversity was observed within X. perforans, with 137 BOX‐PCR haplotypes. Race T3 prevailed (97.5%), but reported here for the first time is the occurrence of five strains identified as race T4 in fresh‐market fields in the state of São Paulo. The race T4 phenotype of these strains resulted from the presence of an 859 bp insertion in the avirulence gene avrXv3. This insertion is related to amino acid sequences of a transposase found in X. gardneri, and to amino acid sequences of X. campestris.     

Monitoring the occurrence of tomato bacterial spot and range of the causal agent Xanthomonas perforans in Iran

A 2‐year comprehensive field survey was conducted across major tomato‐growing areas of Iran. Two hundred and thirty‐four tomato fields and six tomato‐producing greenhouses were surveyed for the potential presence of bacterial spot disease. Five hundred and ninety‐six tomato samples with and without symptoms were analysed. While Xanthomonas spp. were found in association with tomato plants both with and without symptoms from five surveyed counties, the bacterial spot disease was observed only in plants from three of them. Only strains isolated from plants with symptoms induced disease symptoms on tomato, while those isolated from symptomless plants caused symptoms only on cabbage and common bean. None of the isolates caused disease symptoms on pepper and eggplant. Phylogenetic analysis showed that X. perforans is the causal agent of tomato bacterial spot in Iran, although X. campestris and X. axonopodis were also associated with symptomless tomato plants. All X. perforans isolates in this study were sensitive to streptomycin, copper sulphate and copper oxychloride at concentrations of 50 mg L^?1, 200 mg L^?1 and 0.8 g L^?1, respectively. Unlike the type strain of X. perforans, isolates in this study did not produce bacteriocin against other Xanthomonas spp., nor were they detected using the usual species‐specific primer pair Bs‐XpF/Bs‐XpR. This suggests an atypical nature of X. perforans strains in Iran, which leads to the hypothesis that X. perforans strains in Iran may have a separate origin to those causing disease epidemics elsewhere. The aggregated dispersal pattern of the diseased tomato fields signifies the seedborne introduction of the pathogen into the country.     

Screening tomato genotypes for resistance and tolerance to Tomato chlorosis virus

Tomato chlorosis virus (ToCV) is an emerging crinivirus in Brazil that causes an economically important disease in tomato (Solanum lycopersicum) and other solanaceous species. ToCV is transmitted predominantly by the whitefly Bemisia tabaci Middle East‐Asia Minor 1 (MEAM1, formerly biotype B), in a semipersistent manner. As all cultivated tomato varieties and hybrids are susceptible to this crinivirus, the main alternatives for the control of the disease are the use of healthy seedlings for transplanting and the chemical control of the insect vector. The objective of this work was to evaluate the responses of tomato genotypes to infection with this crinivirus and their tolerance to the disease in order to support the development of other alternatives for disease control. Resistance to infection was evaluated by ToCV inoculation with viruliferous B. tabaciMEAM1 followed by virus detection by RT‐PCR and RT‐qPCR. To measure tolerance to the disease, plant development and fruit yield of ToCV‐infected and healthy plants were compared. Among 56 genotypes, only the lineage IAC‐CN‐RT (S. lycopersicum ‘Angela Gigante’ × S. peruvianum ‘LA 444‐1’) was highly resistant to infection with ToCV. Tolerance to the disease over two trials with different genotypes showed variable results. The effect of ToCV on plant development varied from 2.9% to 71.9% reduction, while yield loss varied from 0.2% to 51.8%. The highly ToCV‐resistant lineage IAC‐CN‐RT, which is also resistant to a Spanish isolate of ToCV, might be useful for tomato breeding programmes.     

Assessing host specialization of Botrytis cinerea on lettuce and tomato by genotypic and phenotypic characterization

This study tested the hypothesis that Botyrtis cinerea shows host specialization on tomato and lettuce, using phenotypic and genotypic tools. Strains were isolated from tomato and lettuce grown together in the same greenhouse. Forty‐four lettuce strains and 42 tomato strains were investigated for their genetic diversity and their aggressiveness. Both gene diversity and allelic richness were significantly higher in lettuce strains than in tomato strains (P = 0·01). Cluster analysis revealed a clear division of the strains under study into two clusters. However, this structure did not separate the strains according to their host of origin. Tomato strains were significantly more aggressive than lettuce strains when inoculated on tomatoes (P = 0·001), but no significant differences in aggressiveness were observed when the strains were inoculated on lettuce (P = 0·17) or on apple (P = 0·87). The results suggest an absence of clear host specialization of B. cinerea on tomato and lettuce.     

Identification of an A2 population of Phythophthora andina attacking tree tomato in Peru indicates a risk of sexual reproduction in this pathosystem

Tree tomato, Solanum betaceum, is an Andean fruit crop previously shown to be attacked by Phytophthora andina in Ecuador and Colombia. Blight‐like symptoms were discovered on tree tomato plants in the central highlands of Peru in 2003 and shown to be caused by P. andina. Isolates of P. andina, collected from three different plantations in Peru over a 6‐year time span (2003–2008), were compared genetically with P. andina isolates from Colombia and Ecuador to test whether the pathogen population is geographically structured in the Andes. Restriction fragment length polymorphism (RFLP), mitochondrial DNA and simple sequence repeat (SSR) genetic markers, and mating type behaviour indicated that the Peruvian P. andina population from tree tomato is genetically distinct from populations infecting tree tomato in Colombia (CO‐1) and Ecuador (EC‐3, Ia, A1), but is more similar to the population infecting solanaceous hosts of the Anarrhichomenum complex (EC‐2, Ic, A2). Such geographic substructuring within this pathogen species could result from spatial isolation. Most strikingly, in contrast to the Ecuadorian and Colombian P. andina isolates from tree tomato, the Peruvian isolates have the A2 mating type. The presence of both mating types in the Andean population of P. andina attacking tree tomato indicates a risk of sexual reproduction and the presence of long‐lasting oospores in this pathosystem.     

Biological characterization of Tomato leaf curl New Delhi virus from Spain

Tomato leaf curl New Delhi virus (ToLCNDV; family Geminiviridae, genus Begomovirus) is an emerging virus in horticulture crops in Asia, and has recently been introduced in Spain, Tunisia and Italy. No betasatellite DNA was detected in infected tomato and zucchini squash samples from Spain, and agroinoculated viral DNA‐A and DNA‐B were sufficient to reproduce symptoms in plants of both crop species. Infected tomato and zucchini squash plants also served as inoculum sources for efficient transmission either mechanically or using Bemisia tabaci whiteflies. Cucumber, melon, watermelon, zucchini squash, tomato, eggplant and pepper, but not common bean, were readily infected using viruliferous whiteflies and expressed symptoms 8–15 days post‐inoculation. New full‐length sequences from zucchini squash and tomato indicated a high genetic homogeneity (>99% sequence identity) in the ToLCNDV populations in Spain, pointing to a single recent introduction event.     

Variation of pathotypes and races and their correlations with clonal lineages in Verticillium dahliae

Understanding pathogenic variation in plant pathogen populations is key for the development and use of host resistance for managing verticillium wilt diseases. A highly virulent defoliating (D) pathotype in Verticillium dahliae has previously been shown to occur only in one clonal lineage (lineage 1A). By contrast, no clear association has yet been shown for race 1 with clonal lineages. Race 1 carries the effector gene Ave1 and is avirulent on hosts that carry resistance gene Ve1 or its homologues. The hypothesis tested was that race 1 arose once in a single clonal lineage, which might be expected if V. dahliae acquired Ave1 by horizontal gene transfer from plants, as hypothesized previously. In a diverse sample of 195 V. dahliae isolates from nine clonal lineages, all race 1 isolates were present only in lineage 2A. Conversely, all lineage 2A isolates displayed the race 1 phenotype. Moreover, 900‐bp nucleotide sequences from Ave1 were identical among 27 lineage 2A isolates and identical to sequences from other V. dahliae race 1 isolates in GenBank. The finding of race 1 in a single clonal lineage, with identical Ave1 sequences, is consistent with the hypothesis that race 1 arose once in V. dahliae. Molecular markers and virulence assays also confirmed the well‐established finding that the D pathotype is found only in lineage 1A. Pathogenicity assays indicated that cotton and olive isolates of the D pathotype (lineage 1A) were highly virulent on cotton and olive, but had low virulence on tomato.     

Magnesium oxide nanoparticles induce systemic resistance in tomato against bacterial wilt disease

Bacterial wilt is a serious problem affecting many important food crops. Recent studies have indicated that treatment with biotic or abiotic stress factors may increase the resistance of plants to bacterial infection. This study investigated the effects of magnesium oxide nanoparticles (MgO NP) on disease resistance in tomato plants against Ralstonia solanacearum, as well as its antibacterial activity. The roots of tomato seedlings were inoculated with R. solanacearum and then immediately treated with MgO NP; the treated plants showed very little inhibition of bacterial wilt. In contrast, when roots were drenched with a MgO NP suspension prior to inoculation with the pathogen, the incidence of disease was significantly reduced. Rapid generation of reactive oxygen species such as O₂^?˙ radicals was observed in tomato roots treated with MgO NP. Further O₂^?˙ was rapidly generated when tomato plant extracts or polyphenols were added to the MgO NP suspension, suggesting that the generation of O₂^?˙ in tomato roots might be due to a reaction between MgO NP and polyphenols present in the roots. Salicylic acid‐inducible PR1, jasmonic acid‐inducible LoxA, ethylene‐inducible Osm, and systemic resistance‐related GluA were up‐regulated in both the roots and hypocotyls of tomato plants after treatment of the plant roots with MgO NP. Histochemical analyses showed that β‐1,3‐glucanase and tyloses accumulated in the xylem and apoplast of pith tissues of the hypocotyls after MgO NP treatment. These results indicate that MgO NP induces systemic resistance in tomato plants against R. solanacearum.     

Detection of Tomato yellow leaf curl virus in imported tomato fruit in northern Europe

Imported tomato fruits infected with Tomato yellow leaf curl virus (TYLCV) were identified on the market in northern Europe using paper‐based FTA Classic Cards (Whatman), polymerase chain reaction (PCR) and partial DNA sequence analysis. Trade tomatoes originating from southern Europe, Africa and the Middle East were sampled in Estonia and Sweden, and tested for infection with begomoviruses. Out of 100 batches analysed with five fruits sampled in each batch (58 batches from Estonia and 42 from Sweden), 20 batches were positive (16 from Estonia and four from Sweden). Rolling circle amplification (RCA) and full‐length genome sequence analysis of one isolate collected in Estonia and one isolate in Sweden, revealed highest nucleotide sequence identity at 99% to TYLCV‐IL for the Estonian isolate and at 97% to TYLCV‐Mld for the Swedish isolate. In this study, TYLCV was identified for the first time in imported tomato fruits on the market in northern Europe. FTA cards proved to be an effective means to collect, extract and store begomovirus DNA from tomato fruits and the subsequent molecular analysis.     

Multilocus sequence analysis reveals a novel phylogroup of Xanthomonas euvesicatoria pv. perforans causing bacterial spot of tomato in Iran

A multilocus sequence analysis (MLSA) was performed on five housekeeping genes (fusA, gapA, gltA, lacF and lepA) of 22 Xanthomonas euvesicatoria strains recently isolated from alfalfa, pepper and tomato plants in Iran. In addition, 161 strains isolated worldwide from pepper, poinsettia, rose and tomato plants were included in the analysis. All X. euvesicatoria pv. perforans isolates from tomato plants in Iran clustered in a monophyletic group, although five MLSA haplotypes were detected among them. The Iranian tomato strains presented 10 nucleotide differences in the lepA gene sequences compared to the known worldwide population of X. euvesicatoria pv. perforans. Statistical analyses revealed a recombination event that had occurred in the lepA gene of the strains isolated from tomato in Iran. BOX‐PCR analysis confirmed the inclusion of Iranian tomato strains within X. euvesicatoria pv. perforans. Furthermore, X. euvesicatoria pv. euvesicatoria strains isolated from pepper in Iran differed in one nucleotide in the lepA gene sequence from the known worldwide population of the pathovar, and clustered in a group containing strains isolated in Nigeria. The strains isolated from alfalfa in Iran clustered with the type strain of X. euvesicatoria pv. alfalfae. Altogether, the results reveal the existence of a phylogenetically novel population of X. euvesicatoria pv. perforans in Iran which needs further in‐depth analysis to pinpoint the epidemiological impact of these strains.     

Genetic differentiation and host preference reveal non‐exclusive host races in the generalist parasitic weed Phelipanche ramosa

We developed 20 microsatellite markers to genotype over 100 populations of the parasitic weed Phelipanche ramosa, which covers a wide host crop and geographic range. A representative core collection of 15 populations was also used in cross‐infestation assays to study host preference during germination, attachment and shoot formation. We observed low genetic differentiation within most of the populations, but high genetic differentiation between populations partitioned into 3 genetic groups with different host preferences and geographic distributions. Genetic group 1 is detected exclusively in western France and on various host crops, notably winter oilseed rape (WOSR) and not hemp. Cross‐infection assays confirmed its incompatibility with hemp and showed its preference for WOSR and tobacco in terms of germination and attachment success. The group 2 populations share a large geographic distribution in France and Europe, low germination success with WOSR and high germination success, attachment success and shoot formation with hemp, tobacco or tomato. The subclades 2a and 2b include most of the French populations in hemp crops in eastern France and in tobacco fields in several European countries respectively. The genetic analyses revealed the potential of the three groups to increase their geographic range in the future. Intermediate genetic groups showed higher intrapopulation diversity and represent potential stocks for new host race emergence. Those findings argue in favour of the existence of host races in P. ramosa and should be considered for appropriate management strategies, notably in breeding programmes for resistance against this parasitic weed.     

Resistance to a highly aggressive isolate of Sclerotinia sclerotiorum in a Brassica napus diversity set

Sclerotinia stem rot (SSR) of oilseed rape (OSR, Brassica napus), caused by Sclerotinia sclerotiorum, is a serious problem in the UK and worldwide. As fungicide‐based control approaches are not always reliable, identifying host resistance is a desirable and sustainable approach to disease management. This research initially examined the aggressiveness of 18 Sclerotinia isolates (17 S. sclerotiorum, one S. subarctica) on cultivated representatives of B. rapa, B. oleracea and B. napus using a young plant test. Significant differences were observed between isolates and susceptibility of the brassica crop types, with B. rapa being the most susceptible. Sclerotinia sclerotiorum isolates from crop hosts were more aggressive than those from wild buttercup (Ranunculus acris). Sclerotinia sclerotiorum isolates P7 (pea) and DG4 (buttercup), identified as ‘aggressive’ and ‘weakly aggressive’, respectively, were used to screen 96 B. napus lines for SSR resistance in a young plant test. A subset of 20 lines was further evaluated using the same test and also in a stem inoculation test on flowering plants. A high level of SSR resistance was observed for five lines and, although there was some variability between tests, one winter OSR (line 3, Czech Republic) and one rape kale (line 83, UK) demonstrated consistent resistance. Additionally, one swede (line 69, Norway) showed an outstanding level of resistance in the stem test. Resistant lines also had fewer sclerotia forming in stems. New pre‐breeding material for the production of SSR resistant OSR cultivars relevant to conditions in the UK and Europe has therefore been identified.     

Occurrence of azoxystrobin‐resistant isolates in Passalora fulva,the pathogen of tomato leaf mould disease

Since 2007, serious damage to tomato from leaf mould caused by Passalora fulva has frequently been observed in commercial greenhouses in Gifu Prefecture, Japan. One of the factors relating to this damage was suspected to be a decrease in azoxystrobin sensitivity of the pathogen. Biological and molecular studies were conducted to characterize fungicide resistance. In in vitro sensitivity tests using mycelial homogenate placed on fungicide‐amended medium, the minimum inhibitory concentrations (MIC) of azoxystrobin for mycelial growth of the isolates divided into two ranges, 0.031–0.5 mg L^?1 and 8–32 mg L^?1. Isolates with MICs within the two ranges were considered as sensitive and resistant, respectively, to azoxystrobin because, in in vivo tests, the percentage protection conferred by this fungicide (100 mg a.i. L^?1) against these isolates was 89.7–100% and 4.5–31.1%, respectively. Resistant isolates had a replacement of phenylalanine with leucine at codon 129 (F129L) in cytochrome b. Forty‐five percent of the 271 isolates collected from 63 tomato greenhouses from 2007 to 2008 were resistant to azoxystrobin. In many greenhouses where the isolation frequency of resistant isolates was 80% or more, azoxystrobin had been used twice per crop for approximately 6 years. In 2012, 27% of the 405 isolates collected were resistant to azoxystrobin, and there was a marked difference in the frequency of occurrence of resistant isolates in the field populations between the three locations sampled. The occurrence of azoxystrobin‐resistant P. fulva isolates (F129L mutants) inflicted considerable damage on greenhouse tomatoes.     

Diversity of Avr‐vnt1 and AvrSmira1 effector genes in Polish and Norwegian populations of Phytophthora infestans

The oomycete Phytophthora infestans, the cause of late blight, is one of the most important potato pathogens. During infection, it secretes effector proteins that manipulate host cell function, thus contributing to pathogenicity. This study examines sequence differentiation of two P. infestans effectors from 91 isolates collected in Poland and Norway and five reference isolates. A gene encoding the Avr‐vnt1 effector, recognized by the potato Rpi‐phu1 resistance gene product, is conserved. In contrast, the second effector, AvrSmira1 recognized by Rpi‐Smira1, is highly diverse. Both effectors contain positively selected amino acids. A majority of the polymorphisms and all selected sites are located in the effector C‐terminal region, which is responsible for their function inside host cells. Hence it is concluded that they are associated with a response to diversified target protein or recognition avoidance. Diversification of the AvrSmira1 effector sequences, which existed prior to the large‐scale cultivation of plants containing the Rpi‐Smira1 gene, may reduce the predicted durability of resistance provided by this gene. Although no isolates virulent to plants with the Rpi‐phu1 gene were found, the corresponding Avr‐vnt1 effector has undergone selection, providing evidence for an ongoing ‘arms race’ between the host and pathogen. Both genes remain valuable components for resistance gene pyramiding.     

Panel of real‐time PCRs for the multiplexed detection of two tomato‐infecting begomoviruses and their cognate whitefly vector species

A new approach for the simultaneous identification of the viruses and vectors responsible for tomato yellow leaf curl disease (TYLCD) epidemics is presented. A panel of quantitative multiplexed real‐time PCR assays was developed for the sensitive and reliable detection of Tomato yellow leaf curl virus‐Israel (TYLCV‐IL), Tomato leaf curl virus (ToLCV), Bemisia tabaci Middle East Asia Minor 1 species (MEAM1, B biotype) and B. tabaci Mediterranean species (MED, Q biotype) from either plant or whitefly samples. For quality‐assurance purposes, two internal control assays were included in the assay panel for the co‐amplification of solanaceous plant DNA or B. tabaci DNA. All assays were shown to be specific and reproducible. The multiplexed assays were able to reliably detect as few as 10 plasmid copies of TYLCV‐IL, 100 plasmid copies of ToLCV, 500 fg B. tabaci MEAM1 and 300 fg B. tabaci MED DNA. Evaluated methods for routine testing of field‐collected whiteflies are presented, including protocols for processing B. tabaci captured on yellow sticky traps and for bulking of multiple B. tabaci individuals prior to DNA extraction. This work assembles all of the essential features of a validated and quality‐assured diagnostic method for the identification and discrimination of tomato‐infecting begomovirus and B. tabaci vector species in Australia. This flexible panel of assays will facilitate improved quarantine, biosecurity and disease‐management programmes both in Australia and worldwide.     

Arabidopsis thaliana,an experimental host for tomato yellow leaf curl disease‐associated begomoviruses by agroinoculation and whitefly transmission

Tomato yellow leaf curl disease is one of the most devastating viral diseases affecting tomato crops worldwide. This disease is caused by several begomoviruses (genus Begomovirus, family Geminiviridae), such as Tomato yellow leaf curl virus (TYLCV), that are transmitted in nature by the whitefly vector Bemisia tabaci. An efficient control of this vector‐transmitted disease requires a thorough knowledge of the plant–virus–vector triple interaction. The possibility of using Arabidopsis thaliana as an experimental host would provide the opportunity to use a wide variety of genetic resources and tools to understand interactions that are not feasible in agronomically important hosts. In this study, it is demonstrated that isolates of two strains (Israel, IL and Mild, Mld) of TYLCV can replicate and systemically infect A. thaliana ecotype Columbia plants either by Agrobacterium tumefaciens‐mediated inoculation or through the natural vector Bemisia tabaci. The virus can also be acquired from A. thaliana‐infected plants by B. tabaci and transmitted to either A. thaliana or tomato plants. Therefore, A. thaliana is a suitable host for TYLCV–insect vector–plant host interaction studies. Interestingly, an isolate of the Spain (ES) strain of a related begomovirus, Tomato yellow leaf curl Sardinia virus (TYLCSV‐ES), is unable to infect this ecotype of A. thaliana efficiently. Using infectious chimeric viral clones between TYLCV‐Mld and TYLCSV‐ES, candidate viral factors involved in an efficient infection of A. thaliana were identified.     

Genetic architecture of adult plant resistance to leaf rust in a wheat association mapping panel

Leaf rust caused by Puccina triticina is one of the most destructive fungal diseases of wheat (Triticum aestivum). Adult plant resistance (APR) is an effective strategy to achieve long‐term protection from the disease. In this study, findings are reported from a genome‐wide association study (GWAS) using a panel of 96 wheat cultivars genotyped with 874 Diversity Arrays Technology (DArT) markers and tested for adult leaf rust response in six field trials. A total of 13 quantitative trait loci (QTL) conferring APR to leaf rust were identified on chromosome arms 1BL, 1DS, 2AS, 2BL, 2DS, 3BS, 3BL, 4AL, 6BS (two), 7DS, 5BL/7BS and 6AL/6BS. Of these, seven QTLs mapped close to known resistance genes and QTLs, while the remaining six are novel and can be used as additional sources of resistance. Accessions with a greater number of combined QTLs for APR showed lower levels of disease severity, demonstrating additive and significant pyramiding effects. All QTLs had stable main effects and they did not exhibit a significant interaction with the experiments. These findings could help to achieve adequate levels of durable resistance through marker‐assisted selection and pyramiding resistance QTLs in local germplasm.