Differential interactions of <Emphasis Type="Italic">Verticillium longisporum</Emphasis> and <Emphasis Type="Italic">V. dahliae</Emphasis> with <Emphasis Type="Italic">Brassica napus</Emphasis> detected with molecular and histological techniques

The differential interactions of V. longisporum (VL) and V. dahliae (VD) on the root surface and in the root and shoot vascular system of Brassica napus were studied by confocal laser scanning microscopy (CLSM), using GFP tagging and conventional fluorescence dyes, acid fuchsin and acridin orange. VL and VD transformants expressing sGFP were generated by Agrobacterium-mediated transformation. GFP signals were less homogenous and GFP tagging performed less satisfactory than the conventional fluorescence staining when both were studied with CLSM. Interactions of both pathogens were largely restricted to the root hair zone. At 24 h post-inoculation (hpi), hyphae of VL and VD were found intensely interwoven with the root hairs. Hyphae of VL followed the root hairs towards the root surface. At 36 hpi, VL hyphae started to cover the roots with a hyphal net strictly following the grooves of the junctions of the epidermal cells. VL started to penetrate the root epidermal cells without any conspicuous infection structures. Subsequently, hyphae grew intracellularly and intercellularly through the root cortex towards the central cylinder, without inducing any visible plant responses. Colonisation of the xylem vessels in the shoot with VL was restricted to individual vessels entirely filled with mycelium and conidia, while adjacent vessels remained completely unaffected. This may explain why no wilt symptoms occur in B. napus infected with VL. Elevated amounts of fungal DNA were detectable in the hypocotyls 14 days post-inoculation (dpi) and in the leaves 35 dpi. Root penetration was also observed for VD, however, with no directed root surface growth and mainly an intercellular invasion of the root tissue. In contrast to VL, VD started ample formation of conidia on the roots, and was unable to spread systemically into the shoots. VD did not form microsclerotia in the root tissue as widely observed for VL. This study confirms that VD is non-pathogenic on B. napus and demonstrates that non-host resistance against this fungus materializes in restriction of systemic spread rather than inhibition of penetration.     

Contribution to the taxonomy and pathogenicity of fungicolous Verticillium species. II. Pathogenicity

In recent years in the Netherlands a second mushroom species,Agaricus bitorquis, which prefers higher temperatures thanA. bisporus and is less susceptible to certain diseases, is often commercially grown.Verticillium fungicola var.fungicola, the causal agent of dry bubble, is responsible for considerable damage in crops ofA. bisporus. InA. bitorquis, however, dry bubble has hardly been noticed, but brown spots due toV. fungicola var.aleophilum resulted in inferior mushroom quality. The latter variety also caused brown spots ina. bisporus, but to a minor degree. In variety Les Miz 60 ofA. bisporus, however, it also induced fruit-body deformation in a way different from dry bubble. Verticillium psalliotae, isolated fromA. bitorquis in England, induced more confluent brown spots inA. bitorquis. In the netherlands, where moreA. bitorquis is grown than in other countries,V. psalliotae has not yet been encountered in crops ofA. bitorquis. V. psalliotae, which has a high temperature optimum for mycelial growth, likeV. fungicola var.aleophilum andA. bitorquis, did not infectA. bisporus in our trials.Artificial infection ofA. bisporus orA. bitorquis could not be accomplished with the following related and/or fungicolous fungi:Verticillium lamellicola, V. fungicola var.flavidum, V. biguttatum, Nectriopsis tubariicola, Acremonium crotocinigenum andAphanocladium album.Samenvatting Vooral in Nederland wordt sinds een aantal jaren naastAgaricus bisporus ook de warmteminnende champignonsoortAgaricus bitorquis geteeld, die minder vatbaar is voor bepaalde ziekten. TerwijlVerticillium fungicola varfungicola in de teelt vanA. bisporus droge mollen en daardoor veel schade veroorzaakt, komen in de teelt vanA. bitorquis geen droge mollen voor maar wel bruine vlekken, die tot kwaliteitsverlies en dus schade leiden. De vlekken bleken veroorzaakt te worden doorV. fungicola var.aleophilum. Deze schimmel veroorzaakte ook inA. bisporus bruine vlekken, hoewel in minder ernstige mate, maar in het ras Les Miz 60 vanA. bisporus bovendien misvormde champignons, die wel op droge mollen leken, maar daaraan niet gelijk waren.OokV. psalliotae, in Engeland geïsoleerd vanA. bitorquis met vlekken, veroorzaakte wat meer samenvloeiende, bruine vlekken inA. bitorquis. In Nederland, waar meerA. bitorquis geteeld wordt dan in andere landen, isV. psalliotae nog niet aangetroffen in teelten vanA. bitorquis. InA. bisporus kon geen kunstmatige infectie worden verkregen metV. psalliotae, die net alsV. fungicola var.aleophilum enA. bitorquis warmteminnend genoemd zou kunnen worden.Met de volgendeVerticillium-achtige of van paddestoelen geïsoleerde schimmels kon evenmin op kunstmatige wijze een infectie worden opgeroepen inA. bisporus ofA. bitorquis: Verticillium lamellicola, V. fungicola var.flavidum, V. biguttatum, Nectriopsis tubariicola, Acremonium crotocinigenum enAphanocladium album.     

Assessment of real-time PCR as a method for determining the presence of <Emphasis Type="Italic">Verticillium dahliae</Emphasis> in different Solanaceae cultivars

Real-time PCR was used to detect and quantify Verticillium dahliae and to assess the susceptibility of four Capsicum annuum cultivars (Luesia, Padrón, SCM331 and PI201234) and the Capsicum chinense cv. C118 to this pathogen. The symptoms which developed after infection included stunting and yellowing, and were more acute in the cv. SCM331, which also suffered defoliation in later stages of the disease and in C118, which suffered severe stunting. Quantification of the pathogen DNA in roots 23 and 34 days post-inoculation (dpi) revealed that there were significantly higher amounts of Verticillium dahliae DNA in C118 than in the other cultivars, followed by SCM331, Padrón and PI201234. The lowest amounts of fungal DNA in roots were found in Luesia. In hypocotyls, the highest amounts of fungal DNA were found in SCM331, while Luesia, Padrón and PI201234 had much lower amounts, and C118 had intermediate levels. When a compatible versus an incompatible system was studied, using the near-isogenic tomato lines LA3030 (susceptible) and LA3038 (resistant to V. dahliae), we were able to detect fungal DNA in both lines. As expected, the fungus/plant DNA ratio was lower in LA3038 than in LA3030 and it decreased with time in LA3038. The amount of Verticillium dahliae DNA in the roots of LA3030 remained constant between days 23 and 34 post-inoculation, but increased 10-fold in collars. Finally, when real-time PCR was applied as a diagnostic method to samples from pepper plants, soil and water collected from farms in northwest Spain, we were able to detect V. dahliae DNA in these samples even when symptoms of the disease were not evident.     

<Emphasis Type="Italic">In vitro</Emphasis> screening for sunflower (<Emphasis Type="Italic">Helianthus annuus</Emphasis>L.) resistant calli to <Emphasis Type="Italic">Diaporthe helianthi</Emphasis> fungal culture filtrate

Diaporthe helianthi the causal agent of sunflower (Helianthus annuus) stem canker, causes significant reductions in yield and oil content in most sunflower-growing areas. With the aim of enhancing host resistance, we selected in vitro sunflower calli against culture filtrates of two pathogen isolates (7/96 and 101/96). This technique may be an effective and rapid tool to discriminate the most virulent D. helianthi isolate and to screen for host resistance in the early stage of a breeding programme. Further investigation on the mechanisms involved in defence pathways showed no induction of salicylic acid and pathogenesis-related proteins in calli, indicating that the host resistance is not associated with Systemic Acquired Resistance but probably other biochemical mechanisms.     

Cloning of DNA fragments specific to the pathotype and race of <Emphasis Type="Italic">Verticillium dahliae</Emphasis>

Japanese isolates of Verticillium dahliae, a causal agent of wilt disease in many plants, are classifiable into pathotypes based on their pathogenicity. Because these pathotypes are morphologically indistinguishable, establishing a rapid identification method is very important for the control of this pathogen in Japan. For cloning DNA fragments that are useful for identification and specific detection of V. dahliae pathotypes, we performed random amplified polymorphic DNA (RAPD) analyses using various isolates. One polymerase chain reaction (PCR) product, E10-U48, was specific to isolates pathogenic to sweet pepper. The other product, B68-TV, was specific to race 1 of isolates pathogenic to tomato. The specificity of these sequences was confirmed by genomic Southern hybridization. Further analyses revealed that the region peripheral to B68-TV obtained from the genomic DNA library includes the sequence specific to all isolates pathogenic to tomato (races 1 and 2). Moreover, sequence tagged site (STS) primers designed from B68-TV and its peripheral region showed race-specific and pathotype-specific amplification in a PCR assay. The probes and primers obtained in this study are likely to be useful tools for the identification and specific detection of pathotypes and races of V. dahliae. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession number AB095266.     

Host range expansion in a powdery mildew fungus (<Emphasis Type="Italic">Golovinomyces</Emphasis> sp.) infecting <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>: <Emphasis Type="Italic">Torenia fournieri</Emphasis> as a new host

Since 2003, Torenia fournieri plants grown for experimental purposes were repeatedly infected by powdery mildew in a laboratory in Hungary. Based on morphological characteristics, the pathogen belonged to the mitosporic genus Oidium subgen. Reticuloidium, the anamorph stage of Golovinomyces. The rDNA ITS sequence was identical to that of two other powdery mildew fungi, infecting Arabidopsis and Veronica, respectively, in different parts of the world. According to a previous phylogenetic analysis of ITS and 28S rDNA sequences, those two powdery mildews belong to a recently evolved group of Golovinomyces characterized by multiple host range expansions during their evolution. Both the ITS sequence and the morphological data indicate that the powdery mildew anamorph infecting Torenia also belongs to this group. It is likely that the powdery mildew infections of the experimental T. fournieri plants, native to south-east Asia, were the result of a very recent host range expansion of a polyphagous Golovinomyces because (i) T. fournieri is absent from our region, except as an experimental plant grown in the laboratory, (ii) the powdery mildew fungus infecting this exotic plant belongs to a group of Golovinomyces where host range expansion is a frequent evolutionary scenario, (iii) cross-inoculation tests showed that this pathogen is also able to infect other plant species, notably A. thaliana and tobacco, and (iv) no Golovinomyces species are known to infect T. fournieri anywhere in the world. Although host range expansion has often been proposed as a common evolutionary process in the Erysiphales, and also in other biotrophic plant pathogens, this has not been clearly demonstrated in any case studies so far. To our knowledge, this is the first convincing case of a host range expansion event in the Erysiphales.     

<Emphasis Type="Italic">Berberis</Emphasis> phyllody is a phytoplasma-associated disease

Berberis thunbergii atropurpurea, also known as Red barberry, is a small ornamental shrub in the family Berberidaceae. In recent years, a phyllody symptom has been observed frequently, spreading in the shrubs in northwestern China. A phytoplasma 16S rDNA specific fragment was amplified by PCR from Berberis plants with the phyllody symptoms. DNA sequencing and restriction fragment length polymorphism analysis revealed the phytoplasma belongs to 16SrV-B subgroup. This is the first report that Berbegis thunbergii atropurpurea is a host for 16SrV-B phytoplasma. The disease was named Berberis phyllody.     

Characteristics of a <Emphasis Type="Italic">Plasmopara angustiterminalis</Emphasis> isolate from <Emphasis Type="Italic">Xanthium strumarium</Emphasis>

Leaves of Xanthium strumarium infected with downy mildew were collected in the vicinity of a sunflower field in southern Hungary in 2003. Based on phenotypic characteristics of sporangiophores, sporangia and oospores as well as host preference the pathogen was classified as Plasmopara angustiterminalis. Additional phenotypic characters were investigated such as the size of sporangia, the number of zoospores per sporangium and the time-course of their release. Infection studies revealed infectivity of the P. angustiterminalis isolate to both X. strumarium and Helianthus annuus. Inoculation of the sunflower inbred line, HA-335 with resistance to all known P. halstedii pathotypes, resulted in profuse sporulation on cotyledons and formation of oospores in the bases of hypocotyls. Infections of sunflower differential lines often led to damping-off. Molecular genetic analysis using simple sequence repeat primers and nuclear rDNA sequences revealed clear differences to Plasmopara halstedii, the downy mildew pathogen of sunflower.     

A novel pathosystem to study the interactions between <Emphasis Type="Italic">Lotus japonicus</Emphasis> and <Emphasis Type="Italic">Fusarium solani</Emphasis>

A wilt disease of the model legume Lotus japonicus was observed in a greenhouse in Tokyo, Japan in May 2004. Roots of diseased plants were rotted and dark brown with lesions spreading to lower stems and leaves, resulting in rapid plant death. The causal agent was identified as Fusarium solani based on the morphology. Sequence analysis of rDNA supported the identification. Inoculation of roots of healthy plants with conidia reproduced characteristic disease symptoms, and F. solani was reisolated from lesions, satisfying Koch’s postulates. The isolate also caused chlorotic to necrotic lesions on leaves of healthy plants after wound-inoculation. Infection by F. solani of leaves of L. japonicus was confirmed histologically. Mycelia were observed in the intercellular spaces of parenchymatous tissues in the lesion area and the surrounding tissues. This is the first report of fungal disease on L. japonicus satisfying Koch’s postulates. We named it “Fusarium root rot of L. japonicus” as a new disease. The compatibility of L. japonicus and F. solani is expected to form a novel pathosystem for studying interactions between legumes and fungal pathogens. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AB258993 and AB258994.     

Genetic diversity of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">spinaciae</Emphasis> in Japan based on phylogenetic analyses of rDNA-IGS and <Emphasis Type="Italic">MAT1</Emphasis> sequences

Twenty-eight isolates of Fusarium oxysporum f. sp. spinaciae (FOS; the causal agent of spinach wilt) collected from Japan were assessed for mating type and subjected to phylogenetic analysis. Mating type analysis revealed all isolates to be MAT1-2, suggesting that there is no sexual recombination within the population. Phylogenetic analyses based on nucleotide sequences of the ribosomal DNA intergenic spacer (IGS) and the mating type locus (MAT1) suggested that FOS is polyphyletic. The cluster analysis based on IGS showed four phylogenetic groups (S1–S4) among the isolates. Two distinct lineages, S1 and S3, included FOS isolates both of the vegetative compatibility group (VCG) types, 0330 and 0331, demonstrating that VCG differentiation in FOS may not necessarily reflect the phylogenetic relationships based on IGS and MAT1-2-1.     

Host status and reaction of <Emphasis Type="Italic">Medicago truncatula</Emphasis> accessions to infection by three major pathogens of pea (<Emphasis Type="Italic">Pisum sativum</Emphasis>) and alfalfa (<Emphasis Type="Italic">Medicago sativa</Emphasis>)

Ditylenchus dipsaci, the stem nematode of alfalfa (Medicago sativa), Mycosphaerella pinodes, cause of Ascochyta blight in pea (Pisum sativum) and Aphanomyces euteiches, cause of pea root rot, result in major yield losses in French alfalfa and pea crops. These diseases are difficult to control and the partial resistances currently available are not effective enough. Medicago truncatula, the barrel medic, is the legume model for genetic studies, which should lead to the identification and characterization of new resistance genes for pathogens. We evaluated a collection of 34 accessions of M. truncatula and nine accessions from three other species (two from M. italica, six from M. littoralis and one from M. polymorpha) for resistance to these three major diseases. We developed screening tests, including standard host references, for each pathogen. Most of the accessions tested were resistant to D. dipsaci, with only three accessions classified as susceptible. A very high level of resistance to M. pinodes was observed among the accessions, none of which was susceptible to this pathogen. Conversely, a high level of variation, from resistant to susceptible accessions, was identified in response to infection by A. euteiches.     

Incidence of viruses in <Emphasis Type="Italic">Alstroemeria</Emphasis> plants cultivated in Japan and characterization of <Emphasis Type="Italic">Broad bean wilt virus-2, Cucumber mosaic virus</Emphasis> and <Emphasis Type="Italic">Youcai mosaic virus</Emphasis>

Alstroemeria plants were surveyed for viruses in Japan from 2002 to 2004. Seventy-two Alstroemeria plants were collected from Aichi, Nagano, and Hokkaido prefectures and 54.2% were infected with some species of virus. The predominant virus was Alstroemeria mosaic virus, followed by Tomato spotted wilt virus, Youcai mosaic virus (YoMV), Cucumber mosaic virus (CMV), Alstroemeria virus X and Broad bean wilt virus-2 (BBWV-2). On the basis of nucleotide sequence of the coat protein genes, all four CMV isolates belong to subgroup IA. CMV isolates induced mosaic and/or necrosis on Alstroemeria. YoMV and BBWV-2 were newly identified by traits such as host range, particle morphology, and nucleotide sequence as viruses infecting Alstroemeria. A BBWV-2 isolate also induced mosaic symptoms on Alstroemeria seedlings.     

<Emphasis Type="Italic">Physalis ixocarpa</Emphasis> and <Emphasis Type="Italic">P. peruviana</Emphasis>, new natural hosts of <Emphasis Type="Italic">Tomato chlorosis virus</Emphasis>

Tomato chlorosis virus causes yellow leaf disorder epidemics in many countries worldwide. Plants of Physalis ixocarpa showing abnormal interveinal yellowing and plants of Physalis peruviana showing mild yellowing collected in the vicinity of tomato crops in Portugal were found naturally infected with ToCV. Physalis ixocarpa and P. peruviana were tested for susceptibility to ToCV by inoculation with Bemisia tabaci, Q biotype. Results confirmed that ToCV is readily transmissible to both species. The infection was expressed in P. ixocarpa by conspicuous interveinal yellow areas on leaves that developed into red or brown necrotic flecks, while P. peruviana test plants remained asymptomatic. Infected plants of both P. ixocarpa and P. peruviana served as ToCV sources for tomato infection via B. tabaci transmission. This is the first report of P. ixocarpa and P. peruviana as natural hosts of ToCV.     

Validation of the specificity and sensitivity of species-specific primers that provide a reliable molecular diagnostic for <Emphasis Type="Italic">Xiphinema diversicaudatum</Emphasis>, <Emphasis Type="Italic">X. index</Emphasis> and <Emphasis Type="Italic">X. vuittenezi</Emphasis>

Xiphinema diversicaudatum and X. index are vector nematode species of economic importance in viticulture regions as they can transmit Arabis Mosaic, Grapevine Fanleaf and Strawberry Latent Ringspot viruses to grapevine. Wang et al. (2003) designed species-specific diagnostic primers from ribosomal genes for both these vector species as well as a vector and a non-vector species X. italiae and X. vuittenezi, respectively. Our study aimed to confirm the specificity and determine the sensitivity and reliability of the primers for the two vector species, X. diversicaudatumand X. indexwhen challenged with closely related longidorid species and general nematode communities typical of vineyard soil. With one exception, no PCR product was observed when the primers were tested against six Longidorus, one Paralongidorus and one Xiphinema non-target species. Occasionally (three out of eight replicate PCR reactions) a weak PCR product was noted when primers for X. index were tested with L. elongatus. Furthermore, when challenged with a range of non-target nematode species comprising the nematode community typical of viticulture soil, no PCR product was amplified. An experimental dilution series of extracted DNA rigorously demonstrated that DNA from an equivalent single specimen of the target virus-vector species, X. diversicaudatum and/or X. index, could be detected amongst 1000 equivalent non-targetX. vuittenezi. Also, extracted DNA from an equivalent single target specimen was detected when added to DNA extracted from the overall soil nematode community. The primers were assessed further by using serial mixtures of actual nematodes rather than extracted DNA to simulate field soil. Using this method, a single target nematode could be detected amongst 200 non-target specimens. Given their specificity, sensitivity and reliability, it appears that these diagnostic primers will be of great benefit to phytosanitary/quarantine services related to the viticulture industry.     

Common resistance to different <Emphasis Type="Italic">Fusarium</Emphasis> spp. causing <Emphasis Type="Italic">Fusarium</Emphasis> head blight in wheat

Different sets of wheat genotypes were tested under field conditions by spraying inocula of isolates of seven Fusarium spp. and Microdochium nivale (formerly F. nivale) in the period 1998–2002. The severity of Fusarium head blight (FHB), Fusarium-damaged kernels (FDK), the yield reduction and the deoxynivalenol (DON) contamination were also measured to describe the nature of the resistance. The degrees of FHB severity of genotypes to F. graminearum, F. culmorum, F. avenaceum, F. sporotrichioides, F. poae, F.␣verticillioides, F. sambucinum and M. nivale were very similar, indicating that the resistance to F.␣graminearum was similar to that for other Fusarium spp. listed. This is an important message to breeders as the resistance relates not only to any particular isolate of F. graminearum, but similarly to isolates of other Fusarium spp. This holds true for all the parameters measured. The DON contamination refers only to DON-producers F. graminearum and F. culmorum. Highly significant correlations were found between FHB, FDK, yield loss and DON contamination. Resistance components such as resistance to kernel infection, resistance to DON and tolerance were identified in the more susceptible genotypes. As compared with western European genotypes which produced up to 700 mg kg⁻¹ DON, the Hungarian genotypes produced only 100 mg kg⁻¹ at a similar FDK level. This research demonstrates the importance of measuring both FDK and DON in the breeding and selection of resistant germplasm and cultivars.     

Persistence of <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pv. <Emphasis Type="Italic">vignicola</Emphasis> in weeds and crop debris and identification of <Emphasis Type="Italic">Sphenostylis stenocarpa</Emphasis> as a potential new host

The survival of Xanthomonas axonopodis pv. vignicola, incitant of cowpea bacterial blight and pustule, in residues of infested cowpea leaves was studied in the field in the forest savanna transition zone of South Benin and under variable controlled conditions. The pathogen survived for up to 60 days when placed on the soil surface, and up to 45 days buried at depths of 10 and 20 cm. In the glasshouse, bacteria survived in residue mixed with soil for at least 2 months in dry soil and less than 2 months in moist soil. The pathogen survived at least 30 days in the field after spray-inoculation on the weed species Euphorbia heterophylla, Digitaria horizontalis and Synedrella nodiflora; 20 days on Panicum subalbidum; 10 days on Euphorbia hirta; and 5 days on Talinum triangulare. After leaf-infiltration under glasshouse conditions, the pathogen was detected after 90 days in D. horizontalis; 75 days in T. triangulare, P. subalbidum and S. nodiflora; 60 days in E. hirta, and 30 days in E. heterophylla. Among 12 legume species tested as alternative hosts of X. axonopodis pv. vignicola, only Sphenostylis stenocarpa (African yam bean) showed typical symptoms of cowpea bacterial blight in a glasshouse experiment following artificial inoculation. This is the first time this legume species has been identified as a potential host of X. axonopodis pv.vignicola. Crop residue and weeds are likely sources of primary inoculum when planting two consecutive cowpea crops per year and they probably play a role in dissemination of the pathogen during the cropping season. The alternate host may form a bridge for primary inoculum between cropping seasons.     

Resistance to infection by stealth: <Emphasis Type="Italic">Brassica napus</Emphasis> (winter oilseed rape) and <Emphasis Type="Italic">Pyrenopeziza brassicae</Emphasis> (light leaf spot)

Light leaf spot (Pyrenopeziza brassicae) is an important disease on winter oilseed rape crops (Brassica napus) in northern Europe. In regions where economically damaging epidemics occur, resistance to P. brassicae in commercial cultivars is generally insufficient to control the disease without the use of fungicides. Two major genes for resistance have been identified in seedling experiments, which may operate by decreasing colonisation of B. napus leaf tissues and P. brassicae sporulation. Much of the resistance present in current commercial cultivars is thought to be minor gene-mediated and, in crops, disease escape and tolerance also operate. The subtle strategy of the pathogen means that early colonisation of host tissues is asymptomatic, so a range of techniques and molecular tools is required to investigate mechanisms of resistance. Whilst resistance of new cultivars needs to be assessed in field experiments where they are exposed to populations of P. brassicae under natural conditions, such experiments provide little insight into components of resistance. Genetic components are best assessed in controlled environment experiments with single spore (genetically fixed) P. brassicae isolates. Data for cultivars used in the UK Recommended List trials over several seasons demonstrate how the efficacy of cultivar resistance can be reduced when they are deployed on a widespread scale. There is a need to improve understanding of the components of resistance to P. brassicae to guide the development of breeding and deployment strategies for sustainable management of resistance to P. brassicae in Europe.     

Colonisation and histological changes in muskmelon and autumn squash tissues infected by <Emphasis Type="Italic">Acremonium cucurbitacearum</Emphasis> or <Emphasis Type="Italic">Monosporascus cannonballus</Emphasis>

Muskmelon (Cucumis melo cv. Temprano Rochet) and autumn squash (Cucurbita maxima) seedlings were inoculated either with Acremonium cucurbitacearum or Monosporascus cannonballus, two of the soil-borne fungi implicated in ‘melon collapse’. Inoculation was achieved in two different ways: by growing the plants in pots containing infested soil to study the histological changes produced in the infected tissues using light microscopy and by growing seedlings in Petri dishes together with fungal colonies in order to observe the colonisation of the plant tissues using scanning electron microscopy. Both muskmelon and autumn squash roots infected with A. cucurbitacearum showed a suberised layer in the epidermis and the outermost layers of the parenchymatic cortex, but these symptoms developed earlier in the muskmelon plants. Muskmelon plants infected by this fungus also presented hypertrophy and hyperplasia, which led to a progressive separation of the vascular bundles in the lower stems of the affected plants. This response was not observed in autumn squash during the study. On the other hand, few histological changes were observed in tissues infected with M. cannonballus and only a slight increase in the size of cortical intercellular spaces was noted in the lower stems of muskmelon plants, and infected autumn squash tissues remained free of these symptoms throughout the study. The scanning electron microscope observations revealed that both fungi were able to colonise the tissues of the two host plants which were studied. A. cucurbitacearum colonised the epidermis and cortex of both muskmelon and autumn squash. The hyphae grew both inter- and intracellularly, and the density of the colonisation decreased within the endodermis. The same colonisation of host plants was observed as a result of M. cannonballus infection. The xylem vessel lumina of both muskmelon and autumn squash showed hyphae and tylose formation as a result of both fungal infections. However, non-fungal structures were detected in the hypocotyl vascular tissues. The present study demonstrates that both fungi are capable of infecting the tissues of a species which is resistant (autumn squash) and a species which is susceptible (muskmelon) to melon collapse.     

Isolation of pathogenicity- and gregatin-deficient mutants of <Emphasis Type="Italic">Phialophora gregata</Emphasis> f. sp. <Emphasis Type="Italic">adzukicola</Emphasis> through <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation

Phialophora gregata f. sp. adzukicola, a causal agent of brown stem rot in adzuki beans, produces phytotoxic compounds: gregatins A, B, C, D, and E. Gregatins A, C, and D cause wilting and vascular browning in adzuki beans, which resemble the disease symptoms. Thus, gregatins are considered to be involved in pathogenicity. However, molecular analyses have not been conducted, and little is known about other pathogenic factors. We sought to isolate nonpathogenic and gregatin-deficient mutants through Agrobacterium tumefaciens-mediated transformation (ATMT) for cloning of pathogenicity-related genes. The co-cultivation of P. gregata and A. tumefaciens for 48 h at 20°C with 200 μM acetosyringone resulted in approximately 80 transformants per 10⁶ conidia. The presence of acetosyringone in the A. tumefaciens pre-cultivation period led to an increase in T-DNA copy number per genome. Of 420 and 110 transformants tested for their pathogenicity and productivity of gregatins, one nonpathogenic and three gregatin-deficient mutants were obtained, respectively. The nonpathogenic mutant produced gregatins, whereas the gregatin-deficient mutants had pathogenicity comparable to the wild-type strain. This is the first report of ATMT of P. gregata. Further analysis of these mutants will help reveal the nature of the pathogenicity of this fungus including the role of gregatin in pathogenesis.