Nonspecific toxins as components of a host‐specific culture filtrate from Fusarium oxysporum f. sp. cubense race 1

Bananas and plantains (Musa spp.) are among the most important crops in the world providing staple food for hundreds of millions of people. However, banana production has been devastated by fungal infestations caused by Fusarium oxysporum f. sp. cubense (Foc). Despite the fact that there is very little known on the role of microbial metabolites in the molecular mechanism of Foc infections, it has been proposed that the toxins fusaric acid and beauvericin produced by Foc play an important role during pathogenesis. The aim of this contribution was to study the toxic components of culture filtrates (CF) of Foc and to isolate the extracellular microbial metabolites involved in the plant response. An in vitro bioassay was used to evaluate the production of phytotoxic metabolites as well as the specificity of culture from a strain of Foc belonging to VCG 01210 (race 1). A host‐specific CF was obtained and the phytotoxic compounds characterized as fusaric acid, beauvericin and fumonisin B1. Despite the presence of these nonspecific toxins, a water‐soluble extract from the CF induced protection to the main phytotoxic fraction, measured by lesion area. This hydrophilic fraction induced a fast and strong response of just jasmonic acid (JA)‐dependent defence genes rather than salicylic acid (SA)‐ and ethylene (ET)‐response genes in resistant cultivars. Extracellular proteins isolated from CF of Foc provide an important source for further investigations on the molecular basis of the interaction between Foc and banana.     

A new aubergine disease caused by a whitefly‐borne strain of Tomato mild mottle virus (TomMMoV)

The aims of the present study were to further characterize the causal agent of a new viral disease of aubergines in Israel, first observed in 2003 and tentatively named eggplant mild leaf mottle virus (EMLMV) in a previous work, and to identify the vector responsible for its spread. The disease could be transmitted mechanically from infected source plants to healthy aubergines or laboratory test plants. Transmission electron microscopy (TEM) analysis of purified virus preparations indicated the presence of viral particles with a flexible filamentous morphology (approximately 720 nm long). TEM analysis of ultrathin sections prepared from infected leaf tissue revealed the presence of cytoplasmic inclusion bodies with pinwheel and crystalline structures, typical of those induced by potyviral infection. The viral coat protein subunit was shown to have a molecular weight of 37·5 kDa by SDS‐PAGE analysis. The viral particles reacted positively in western blot analysis with an antiserum against Tomato mild mottle virus (TomMMoV) from Yemen, described as a potyvirus, vectored by the aphid Myzus persicae. The current study describes some biological properties of EMLMV and presents evidence for its transmission by the whitefly Bemisia tabaci, but not by three aphid species. The taxonomic relationship between EMLMV and TomMMoV is discussed based on their biological characteristics and sequence analysis of their genomes. It is suggested that the Israeli EMLMV should be considered a distant strain of TomMMoV, designated TomMMoV‐IL, according to the present rules of Potyviridae molecular taxonomy.     

Colonization of lettuce cultivars and rotation crops by Fusarium oxysporum f. sp. lactucae,the cause of fusarium wilt of lettuce

The severity of fusarium wilt is affected by inoculum density in soil, which is expected to decline during intervals when a non‐susceptible crop is grown. However, the anticipated benefits of crop rotation may not be realized if the pathogen can colonize and produce inoculum on a resistant cultivar or rotation crop. The present study documented colonization of roots of broccoli, cauliflower and spinach by Fusarium oxysporum f. sp. lactucae, the cause of fusarium wilt of lettuce. The frequency of infection was significantly lower on all three rotation crops than on a susceptible lettuce cultivar, and the pathogen was restricted to the cortex of roots of broccoli. However, F. oxysporum f. sp. lactucae was isolated from the root vascular stele of 7·4% of cauliflower plants and 50% of spinach plants that were sampled, indicating a greater potential for colonization and production of inoculum on these crops. The pathogen was also recovered from the root vascular stele of five fusarium wilt‐resistant lettuce cultivars. Thus, disease‐resistant plants may support growth of the pathogen and thereby contribute to an increase in soil inoculum density. Cultivars that were indistinguishable based on above‐ground symptoms, differed significantly in the extent to which they were colonized by F. oxysporum f. sp. lactucae. Less extensively colonized cultivars may prove to be superior sources of resistance to fusarium wilt for use in breeding programmes.     

Role of co‐infection by Pectobacterium and Verticillium dahliae in the development of early dying and aerial stem rot of Russet Burbank potato

Potato early dying (PED) is a disease complex primarily caused by the fungus Verticillium dahliae. Pectolytic bacteria in the genus Pectobacterium can also cause PED symptoms as well as aerial stem rot (ASR) of potato. Both pathogens can be present in potato production settings, but it is not entirely clear if additive or synergistic interactions occur during co‐infection of potato. The objective of this study was to determine if co‐infection by V. dahliae and Pectobacterium results in greater PED or ASR severity using a greenhouse assay and quantitative real‐time PCR to quantify pathogen levels in planta. PED symptoms caused by Pectobacterium carotovorum subsp. carotovorum isolate Ec101 or V. dahliae isolate 653 alone included wilt, chlorosis and senescence and were nearly indistinguishable. Pectobacterium wasabiae isolate PwO405 caused ASR symptoms including water‐soaked lesions and necrosis. Greater Pectobacterium levels were detected in plants inoculated with PwO405 compared to Ec101, suggesting that ASR can result in high Pectobacterium populations in potato stems. Significant additive or synergistic effects were not observed following co‐inoculation with these strains of V. dahliae and Pectobacterium. However, infection coefficients of V. dahliae and Ec101 were higher and premature senescence was greater in plants co‐inoculated with both pathogens compared to either pathogen alone in both trials, and V. dahliae levels were greater in basal stems of plants co‐inoculated with either Pectobacterium isolate. Overall, these results indicate that although co‐infection by Pectobacterium and V. dahliae does not always result in significant additive or synergistic interactions in potato, co‐infection can increase PED severity.     

Evaluation of Malaysian oil palm progenies for susceptibility,resistance or tolerance to Fusarium oxysporum f. sp. elaeidis and defence‐related gene expression in roots

Vascular wilt of oil palm caused by Fusarium oxysporum f. sp. elaeidis (Foe) is a devastating disease in West and Central Africa. As the oil palm industry in southeast Asia is still expanding, so is the oil palm germplasm collection through the importation of seed and pollen from Africa, the centre of diversity for Elaeis guineensis. There is a risk of inadvertent spread of the disease on contaminated seed or pollen. Regular re‐evaluation of the reaction of currently grown palm genotypes towards Foe is clearly required for biosecurity. This study has demonstrated that four Malaysian oil palm progenies, three in current or recent commercial use, are highly susceptible to infection by at least one of two African isolates of Foe, representing different countries, aggressiveness and vegetative compatibility groups. Symptoms and reduction of palm growth generally reflected the extent and intensity of systemic colonization by Foe. Progeny PK 5463 expressed partial resistance to Foe isolate F3, but not to isolate 16F, displaying significantly milder symptoms and supporting less widespread vascular colonization. This relatively incompatible interaction was used to study expression of potential defence‐related genes during root infection when compared to a susceptible palm–isolate combination. The only significant response was an early up‐regulation of chitinase in resistant palms. The research revealed at least one progeny–isolate differential interaction, and the associated resistance expression suggests a component of tolerance, because colonization by Foe was systemic in both compatible and incompatible combinations.     

A new race of Fusarium oxysporum f. sp. lactucae of lettuce

Fusarium oxysporum f. sp. lactucae, the causal agent of fusarium wilt of lettuce (Lactuca sativa), occurs in most countries in which lettuce is grown and causes serious economic losses. Three races (1, 2 and 3) of the pathogen have previously been identified on the basis of their ability to cause disease on differential lettuce cultivars, as well as by means of molecular tools developed to characterize different races of this pathogen. Only race 1 has been detected in Europe so far. In this study, two isolates of F. oxysporum, obtained from lettuce plants grown in the Netherlands showing symptoms of wilt, have been characterized by combining the study of pathogenicity with differential cultivars of lettuce and molecular assays to determine whether the isolates are different from the known races of F. oxysporum f. sp. lactucae. This study reports the presence of F. oxysporum f. sp. lactucae for the first time in the Netherlands. The causal pathogen has been identified, using the IRAP‐SCAR technique, as a new race of F. oxysporum f. sp. lactucae. Specific primers have been designed to identify this new race.     

Rapid detection of Fusarium oxysporum f. sp. lactucae on soil,lettuce seeds and plants using loop‐mediated isothermal amplification

Fusarium oxysporum f. sp. lactucae (FOL) is a soil‐ and seedborne pathogen and the causal agent of fusarium wilt on lettuce. Four races have been identified within FOL, with different worldwide distribution. Several molecular techniques have been used to detect and identify this pathogen; however, not all of them have the optimal characteristics in terms of sensitivity to perform FOL detection in plant and seed material. A loop‐mediated isothermal amplification (LAMP) assay was developed based on the sequence‐characterized amplified region (SCAR) obtained in a previous rapid amplification of polymorphic DNA (RAPD) study. The LAMP assay has been validated according to the EPPO standard PM7/98. The LAMP assay was tested with lettuce seeds, soil and plant material, and can be used successfully to amplify DNA from each of these matrices. In seed lots artificially inoculated with FOL, the detection limit of the LAMP test was 0.004% infected seed.     

Stem rot of jewel orchids caused by a new forma specialis,Fusarium oxysporum f. sp. anoectochili in Taiwan

Stem rot of Anoectochilus formosanus (Af) caused by Fusarium oxysporum (Fo) is a major limiting factor to jewel orchid production in Taiwan. Fo causes discoloration in vascular tissues. However, some newly collected Fo isolates from Af stem rot do not cause vascular discoloration, suggesting changes may have occurred in the pathogen. Among recent Fo isolates from Af there are two colony types, the cottony alba (CA) and the sporodochial (S). In order to confirm that both colony types cause Af stem rot, 200 isolates were obtained from diseased stems in Nantou County and characterized by colony type and whether or not the infected plants had vascular discoloration. Isolates of both the CA and S types caused stem rot of Af; some isolates in each colony type caused vascular discoloration whilst others did not. Pathogenicity tests with 22 isolates resulted in stem rot disease severity ratings on Af of 3·1–4·0 and 2·1–4·0 with CA and S type colonies, respectively. The same isolates failed to cause disease on Cattleya, Dendrobium or Phalaenopsis plants. Phylogenetic analysis of partial intergenic spacer sequences showed that these isolates were distinguishable from other formae speciales of Fo and could be separated into two groups correlated with the CA or S type colonies with high bootstrap. Based on pathogenic, morphological and molecular characterizations, the Fo that causes stem rot of Af is proposed to be a new forma specialis, F. oxysporum f. sp. anoectochili, with different pathotypes.     

Fusarium oxysporum f. sp. radicis‐vanillae is the causal agent of root and stem rot of vanilla

Root and stem rot (RSR) is a very detrimental disease of vanilla worldwide. Fusarium oxysporum is frequently associated with the disease but other Fusarium species are also reported. In this international study, 52 vanilla plots were surveyed in three of the most important vanilla producing countries (Madagascar, Reunion Island and French Polynesia) in order to determine the aetiology of RSR disease. Subsets from the 377 single‐spored Fusarium isolates recovered from rotten roots and stems in the surveys were characterized by molecular genotyping (EF1α and IGS gene sequences) and pathogenicity assays on Vanilla planifolia and V. ×tahitensis, the two commercially grown vanilla species. Fusarium oxysporum was shown to be the principal species responsible for the disease, representing 79% of the isolates recovered from the RSR tissues, 40% of which induced severe symptoms on inoculated plantlets. Fusarium oxysporum isolates were highly polyphyletic regardless of geographic origin or pathogenicity. Fusarium solani, found in 15% of the samples and inducing only mild symptoms on plantlets, was considered a secondary pathogen of vanilla. Three additional Fusarium species were occasionally isolated in the study (F. proliferatum, F. concentricum and F. mangiferae) but were nonpathogenic. Histopathological preparations observed in wide field and multiphoton microscopy showed that F. oxysporum penetrated the root hair region of roots, then invaded the cortical cells where it induced necrosis in both V. planifolia and V. ×tahitensis. The hyphae never invaded the root vascular system up to 9 days post‐inoculation. As a whole, the data demonstrated that RSR of vanilla is present worldwide and that its causal agent should be named F. oxysporum f. sp. radicis‐vanillae.     

Real‐time quantitative PCR assays for the rapid detection and quantification of Fusarium oxysporum f. sp. phaseoli in Phaseolus vulgaris (common bean) seeds

Fusarium oxysporum f. sp. phaseoli (Fop) is a devastating pathogen that can cause significant economic losses and can be introduced into fields through infested Phaseolus vulgaris (common bean) seeds. Efficient seed health testing methods can aid in preventing long‐distance dissemination of this pathogen by contaminated seeds. In order to improve detection of Fop in seed, a rapid, accurate and sensitive real‐time PCR assay (qPCR) protocol was developed for detection of Fop in common bean seeds. Seed lots of seven cultivars with infection incidence ranging from 0·25 to 20% were prepared by mixing known amounts of Fop‐infected seeds with Fop‐free seeds. Direct comparisons between SYBR Green and TaqMan qPCR methods were performed using primers based on the Fop virulence factor ftf1. The primers developed in this study produced a 63 bp product for highly virulent strains of Fop but did not produce an amplicon for nonpathogenic or weakly pathogenic isolates of F. oxysporum from P. vulgaris or other hosts. Under optimized conditions, both qPCR assays detected Fop infection at low levels (0·25%); however, the results suggest the TaqMan assay was more reliable at quantification than the SYBR Green assay. Linear regression models were fitted to the relationships between results of qPCR assays and infection incidence, but the models differed among cultivars. Fungal biomass per seed differed among cultivars and was related to seed size. The results indicate that the TaqMan assay developed in this study is a useful tool for the detection and quantification of Fop in bean seeds.     

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.     

Temperature adaptation in Australasian populations of Puccinia striiformis f. sp. tritici

Stripe rust, caused by Puccinia striiformis f. sp. tritici, is one of the major fungal pathogens of wheat. A new pathotype was introduced to Australia in 2002 and several derivative pathotypes were detected in subsequent seasons. It has been suggested that the severity of stripe rust outbreaks in Australia since 2002 could be as a result of traits other than virulence in the pathogen population. This study was conducted to investigate the hypothesis that the stripe rust pathogen population dominant in Australia since 2002 was better adapted to warm temperature conditions compared to previous pathogen populations. Sixteen pathotypes were selected to examine the influence of two contrasting temperature regimes during the 24 h incubation (10°C and 15°C) and the subsequent post‐inoculation (17°C and 23°C) periods on latent period and infection efficiency on four susceptible wheat cultivars. In addition, the effect of two contrasting incubation temperatures on urediniospore germination was examined. The results indicated that pathotypes of P. striiformis f. sp. tritici detected after 2002 did not show evidence of adaptation to high temperatures, which suggests that other factors contributed to the observed increased aggressiveness.     

利用RAPD-STS和实时定量PCR鉴别菠菜枯萎病病原菌(Fusarium oxysporum f.sp.spinaciae)

 菠菜枯萎病是由致病性镰刀菌(F.o.f.sp.spinaciae)引起的,是菠菜生产中的重要病害之一。利用常规方法鉴定菠菜枯萎病病原菌需耗费大量时间,并且很难得到正确的结论。随机扩增多态性DNA序列标签(Randomly amplified polymorphic DNA-sequence tagged sites,RAPD-STS)为病原菌鉴定提供了一种有效方法。本研究通过对供试菌株的RAPD分析,克隆出了1个菠菜枯萎病病原菌的特异片段(GenBank登录号:AY337463)。根据测序结果设计了1对菠菜枯萎病病原菌的特异引物,并利用常规PCR和实时定量PCR(real-time PCR)2种方法对病原菌进行了鉴定,并对2种方法的敏感性进行了比较。结果表明,2种PCR方法都可以鉴定菠菜枯萎病病原菌(F.o.f.sp.spinaciae),但二者对病原菌DNA敏感程度不同,常规PCR检测的最低DNA量100Pg,而实时定量PCR检测的最低DNA量是1pg。同时,实时定量PCR还可以对病原菌DNA进行定量分析,并依此估算病原菌的数量。该方法可用于菠菜枯萎病病原菌的快速鉴定和病因诊断。     

Pisatin involvement in the variation of inhibition of Fusarium oxysporum f. sp. pisi spore germination by root exudates of Pisum spp. germplasm

Fusarium wilt caused by Fusarium oxysporum f. sp. pisi (Fop) is one of the major constraints of pea worldwide. Its control is difficult and is mainly based on the use of resistant cultivars. This study aimed to identify and characterize resistance mechanisms interfering with Fop spore germination, as an additional pre‐penetration resistance mechanism little explored so far. For this, root exudates were collected from 12 pea accessions with differential responses to the disease, from resistant to susceptible, and their effects on Fop germination and growth were determined. While root exudates from most accessions stimulated Fop germination, the root exudates of three accessions, JI 1412, JI 2480 and P42, did not stimulate, or even inhibited, Fop germination. Although some additional compounds might be involved, the analysis showed that the most active metabolite was the pea phytoalexin pisatin. Pisatin was identified in the active fraction of pea root exudate extracts and its amount in the root exudates was negatively correlated with the extent of Fop germination. This suggests an important role of pisatin in the constitutive defence of pea against F. oxysporum.     

Development of a Fusarium oxysporum f. sp. melonis functional GFP fluorescence tool to assist melon resistance breeding programmes

Fusarium wilt, caused by Fusarium oxysporum f. sp. melonis (Fom), is one of the most widespread and devastating melon diseases. This vascular disease is caused by the colonization of melon xylem vessels by any of the four Fom races reported (r0, r1, r2 and r1,2, subdivided into r1,2w and r1,2y). The macroscopic evaluation of disease symptoms (disease rating, DR) at several days post‐inoculation (dpi) with Fom spores has been the traditional method to determine the resistance of melon accessions to this fungal pathogen. In this study, one isolate from each Fom race was transformed by Agrobacterium tumefaciens to constitutively express the green fluorescent protein (GFP). Fom‐GFP transformants, as virulent as the corresponding wildtype races, were selected to develop an inoculation assay based on the non‐invasive evaluation of the fluorescence emitted by Fom‐GFP. It was determined that melon root neck was the appropriate area to follow Fom‐GFP and a fluorescence signal rating (FSR) was established in parallel to DR determination. This method allowed the evaluation of GFP signal in the root neck of inoculated melon seedlings at 11–15 dpi. The GFP signal was scored in 62 melon accessions/breeding lines inoculated with different Fom‐GFP, followed by evaluation of the macroscopic DR in the aerial part of melon seedlings at 20–28 dpi. Correlation analysis demonstrated a direct and significant relationship between FSR and DR. This method has shown to be an effective and reliable tool that can assist Fom resistance breeding programmes in melon.     

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.     

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.     

Genetic analysis and molecular mapping of resistance to Puccinia striiformis f. sp. pseudo‐hordei in common wheat

This is the first genetic study reporting on the interaction and molecular mapping of resistance to the barley grass stripe rust pathogen (Puccinia striiformis f. sp. pseudo‐hordei, Psph) in common wheat. Seedlings of 638 wheat accessions were tested and it was determined that wheat is a near‐nonhost to Psph based on rare susceptibility observed in <2% of commercial cultivars and <5% of wheat landraces. As previously observed for P. striiformis f. sp. tritici (Pst), the Australian cultivar Teal was highly susceptible to Psph. In contrast, a selection of cv. Avocet carrying complementary resistance genes Yr73 and Yr74 (Avocet R; AvR) was resistant. The Teal × AvR (T/A) doubled haploid (DH) population was used to map resistance in AvR to Psph. Infection types on the T/A DH lines inoculated with Psph and Pst indicated that all DH lines carrying both Yr73 and Yr74 were also resistant to Psph; however, fewer DH lines were susceptible to Psph than expected, suggesting the resistance was more complex. QTL analysis using 9053 DArT‐Seq markers determined that resistance to Psph was polygenically inherited and mapped to chromosomes 3A, 3D, 4A and 5B. The 3DL and 5BL markers co‐located with Yr73 and Yr74, suggesting an overlap between host and non‐host resistance mechanisms.