Analysis of head and leaf reaction towards <Emphasis Type="Italic">Microdochium nivale</Emphasis>

This research examined the variation in the response of eight commercial wheat cultivars to Microdochium nivale isolates using both in vivo FHB tests (AUDPC and RHW measurements) and in vitro detached leaf assays (LGR). Irrespective of fungal variety, the two Italian cvs Fortore and Norba exhibited the greatest amount of visual disease symptoms (mean AUDPC=2.2 and 2.3, respectively), being significantly more susceptible than the other six cultivars (AUDPC 1.24) (P < 0.05). Irrespective of fungal variety, the Italian cv. Norba and the Irish cv. Falstaff were more susceptible than the other cultivars (except Fatima 2) in terms of RHW (P < 0.05), while the cvs Fortore, GK Othalom and Consort were more resistant than the other five cultivars (P < 0.05). In the detached leaf assay, the Hungarian cv. GK Othalom and the Italian cv. Norba were more susceptible (mean LGR=0.79 and 0.81 mm day^–1, respectively) to M. nivalethan the other six cultivars (mean LGR=0.51–0.72) (P < 0.05). Analysis of the relationship between head and leaf reaction to M. nivaleinfection revealed no significant correlation.     

Analysis of protease activity in <Emphasis Type="Italic">Aspergillus flavus</Emphasis> and <Emphasis Type="Italic">A. parasiticus</Emphasis> on peanut seed infection and aflatoxin contamination

Aspergillus flavus and A. parasiticus are aflatoxin-producing fungi that can infect peanut seeds in field crops. An association between A. parasiticus proteolytic enzyme activities and peanut fungal infection was examined. For this study, a model of inductive and non-inductive culture media to produce A. parasiticus extracellular protease before infection was used. These A. parasiticus cultures were used to infect peanut seeds of cultivars resistant and susceptible to aflatoxin contamination. Peanut seeds of both cultivars exposed to fungi grown on casein medium (inductive medium) showed higher internal and external infection and a higher fungal protease content than those observed on potato dextrose agar (PDA) and sucrose medium (non-inductive media). A further study showed higher fungal colonisation and aflatoxin contamination in seeds of the resistant cultivar pre-incubated with Aspergillus extracellular proteases than in those incubated without proteases. Moreover, protease activities affected the viability of non-infected resistant cultivar seeds, inhibiting germination and radicle elongation and enhancing seed tissue injury. The results strongly suggest that protease production by A. parasiticus is involved in peanut seed infection and aflatoxin contamination resulting in seed tissue damage, affecting seed viability and facilitating the access of fungi through the testa. The analysis of fungal extracellular proteases formed on peanut seed during infection showed that A. flavus and A. parasiticus produced metallo and serine proteases; however, there were differences in the molecular masses of the enzymes between both species. The greatest activity in both species was by serine protease, that could be classified as subtilase.     

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.     

Visualisation of <Emphasis Type="Italic">hrp</Emphasis> gene expression in <Emphasis Type="Italic">Xanthomonas euvesicatoria</Emphasis> in the tomato phyllosphere

The plasmid pUFZ75 conferred constitutive GFP expression on the bacterial pathogen Xanthomonas euvesicatoria (syn. X. campestris pv. vesicatoria). Colonisation of the tomato phyllosphere and invasion of tomato leaves by X. euvesicatoria was examined using both fluorescence and confocal laser scanning microscopy. Xanthomonas euvesicatoria established a limited population on the tomato leaf surface, primarily occupying the depressions between epidermal cells and around the stomata, prior to invasion of the leaf via the stomata and subsequent growth within the substomatal chamber and the leaf apoplast. Additionally, hrp-gfp fusions were used to report on the temporal and spatial expression of hrp genes during epiphytic colonisation and invasion. Xanthomonas euvesicatoria cells carrying hrpG- and hrpX-gfp reporter constructs were not fluorescent in vitro on non-hrp-inducing LB agar but did exhibit a low level of fluorescence on the leaf surface within 24 h of inoculation, particularly in the vicinity of stomata. Cells carrying hrpG- and hrpX-gfp fusions exhibited high levels of fluorescence 72 h after inoculation in the substomatal chamber and the leaf apoplast. Cells carrying the hrpF-gfp fusion were slightly fluorescent on LB agar and showed no further increase in fluorescence on the leaf surface by 24 h after inoculation, but did show a significant increase in fluorescence 72 h after inoculation in the substomatal chamber and apoplast. The apparent low-level induction of the regulators hrpG and hrpX on the tomato leaf surface may suggest that some of the genes of the X. euvesicatoria HrpG/HrpX regulon are up- or down-regulated prior to invasion of the stomata while still on the leaf surface.     

The use of GFP<Emphasis Type="Italic">-</Emphasis>transformed isolates to study infection of banana with <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">cubense</Emphasis> race 4

Fusarium oxysporum f. sp. cubense (Foc) is the causal pathogen of Fusarium wilt of banana. To understand infection of banana roots by Foc race 4, we developed a green fluorescent protein (GFP)-tagged transformant and studied pathogenesis using fluorescence microscopy and confocal laser scanning microscopy. The transformation was efficient, and GFP expression was stable for at least six subcultures with fluorescence clearly visible in both hyphae and spores. The transformed Foc isolate also retained its pathogenicity and growth pattern, which was similar to that of the wild type. The study showed that: (i) Foc race 4 was capable of invading the epidermal cells of banana roots directly; (ii) potential invasion sites include epidermal cells of root caps and elongation zone, and natural wounds in the lateral root base; (iii) in banana roots, fungal hyphae were able to penetrate cell walls directly to grow inside and outside cells; and (iv) fungal spores were produced in the root system and rhizome. To better understand the interaction between Foc race 4 and bananas, nine banana cultivars were inoculated with the GFP-transformed pathogen. Root exudates from these cultivars were collected and their effect on conidia of the GFP-tagged Foc race 4 was determined. Our results showed that roots of the Foc race 4-susceptible banana plants were well colonized with the pathogen, but not those of the Foc race 4-resistant cultivars. Root exudates from highly resistant cultivars inhibited the germination and growth of the Fusarium wilt pathogen; those of moderately resistant cultivars reduced spore germination and hyphal growth, whereas the susceptible cultivars did not affect fungal germination and growth. The results of this work demonstrated that GFP-tagged Foc race 4 isolates are an effective tool to study plant–fungus interactions that could potentially be used for evaluating resistance in banana to Foc race 4 by means of root colonization studies. Banana root exudates could potentially also be used to identify cultivars in the Chinese Banana Germplasm Collection with resistance to the Fusarium wilt pathogen.     

Germination and invasion by ascospores and pycnidiospores of <Emphasis Type="Italic">Leptosphaeria maculans</Emphasis> on spring-type <Emphasis Type="Italic">Brassica napus</Emphasis> canola varieties with varying susceptibility to blackleg

The infection processes of ascospores and pycnidiospores of Leptosphaeria maculans were studied on cotyledons of six cultivars of spring-type Brassica napus: one with resistance controlled by a single dominant gene (cv. Surpass 400), three with polygenic resistance (cvs. Dunkeld, Grouse, and Outback), and two susceptible cultivars (Westar and Q2). On all cultivars, ascospore germination, penetration, and development of symptoms on cotyledons were much earlier than that with pycnidiospores. At 2h after inoculation ascospores began to germinate, by 4h about 50% had germinated, and by 6–8h 85%–90% had germinated. In contrast, pycnidiospores began to germinate 1 day after inoculation (dai) and reached only 50% germination by 3 dai. Ascospores began germinating from terminal cells and then later from the interstitial cells. Pycnidiospores germinated predominantly from one end and sometimes from both ends. Germ tubes from ascospores penetrated stomata as early as 4h after inoculation, whereas those from pycnidiospores penetrated at 2 dai. Symptom development with ascospores was 2 days earlier than that with pycnidiospores. Symptoms on Surpass 400 were evident as early as 3–5 dai with ascospores and 5–7 dai with pycnidiospores. However, on other cultivars, symptoms were not evident until 10 dai with ascospores and 12 dai with pycnidiospores. This report is the first on differences in the infection processes by the two spore types. Ascospore and pycnidiospore attachment, germination, and penetration did not differ between resistant and susceptible cultivars, but there were major differences after penetration. Under high humidity, 80%–90% of stomata of susceptible Westar and Q2 had aerial hyphae emerging from stomatal pores. However, fewer stomata (5%–10%) had aerial hyphae on Surpass 400 by 10 dai with ascospores and 12 dai with pycnidiospores, but even these were usually poorly developed. Host differences in spring-type B. napus in relation to production of aerial hyphae have not previously been reported. In Surpass 400, rapid necrosis of guard cells occurred within a few hours of penetration by either type of spore, and subsequently one or a few cells immediately adjacent to the penetration site died. This necrosis then spread to the cells around the penetration site to form a hypersensitive response (in the form of a small, dark lesion) to both ascospores and pycnidiospores. This is the first detailed report on interactions between spring-type B. napus and L. maculans in relation to single dominant gene-based resistance. Neither the cultivars with polygenic resistance nor the susceptible cultivars had such a response.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation for random insertional mutagenesis in <Emphasis Type="Italic">Colletotrichum lagenarium</Emphasis>

Random insertional mutagenesis using a marker DNA fragment is an effective method for identifying fungal genes relevant to morphogenesis, metabolism, and so on. Agrobacterium tumefaciens-mediated transformation (AtMT) has long been used as a tool for the genetic modification of a wide range of plant species. Recent study has indicated that A. tumefaciens could transfer T-DNA not only to plant cells but also to fungal cells. In this study, AtMT was applied to Colletotrichum lagenarium for random insertional mutagenesis. We constructed a binary vector pBIG2RHPH2 carrying a hygromycin-resistant gene cassette between the right and left borders of T-DNA. Optimal co-cultivation of C. lagenarium wild-type 104-T with pBIG2RHPH2-introduced A. tumefaciens C58C1 led to the production of 150–300 hygromycin-resistant transformants per 10⁶ conidia. Southern blot analysis revealed that T-DNA was mainly integrated at a single site in the genome and at different sites in transformants. The T-DNA inserts showed small truncations of either end, but the hygromycin-resistant gene cassette inside the T-DNA was generally intact. The mode of T-DNA insertion described above resulted in highly efficient gene recovery from the transformants by thermal asymmetrical interlaced-polymerase chain reaction. The fungal genomic DNA segments flanking T-DNA were identified from five of eight mutants that had defective melanin biosynthesis. The sequence from one of the segments was identical to that of the melanin biosynthesis gene PKS1 of C. lagenarium, which we previously characterized. These results strongly support our notion that AtMT is a possible tool for tagging genes relevant to pathogenicity in the plant pathogenic fungus C. lagenarium.     

Interactions Between <Emphasis Type="Italic">Barley yellow dwarf virus</Emphasis> and <Emphasis Type="Italic">Fusarium</Emphasis> spp. Affecting Development of Fusarium Head Blight of Wheat

Interactions between Barley yellow dwarf virus (BYDV) and Fusarium species causing Fusarium head blight (FHB) in winter wheat cvs Agent (susceptible to FHB) and Petrus (moderately resistant to FHB) were studied over three years (2001–2003) in outdoor pot experiments. FHB developed more rapidly in cv. Agent than in cv. Petrus. The spread of FHB was greater in BYDV-infected plants than in BYDV-free plants. Thousand grain weight (TGW) was reduced more in Fusarium-infected heads of cv. Agent than in cv. Petrus. A highly significant negative correlation was found between disease index and TGW in cv. Agent (r = −0.916), while in cv. Petrus the correlation was less significant (r = −0.765). Virus infection reduced TGW in cv. Petrus more than in cv. Agent. In plants with both infections, TGW reductions in cv. Petrus corresponded to those of BYDV infection, and in cv. Agent TGW was more diminished than in BYDV infection. Effects of different treatments determined over three years on ergosterol contents in grain were generally similar to effects on disease indices. Grain weight per ear and ear weight of the different treatments of both cultivars largely corresponded with the TGW results. Deoxynivalenol (DON) content in grain of cv. Agent infected with Fusarium spp. was 11–25 times higher compared to the corresponding treatments in cv. Petrus. The DON content in grain of plants of the two cultivars infected with both pathogens was higher than that of plants infected only with Fusarium over the three years.     

Host range and phytotoxicity of <Emphasis Type="Italic">Stemphylium solani</Emphasis>, causing leaf blight of garlic (<Emphasis Type="Italic">Allium sativum</Emphasis>) in China

Since 2004, a new leaf blight disease on garlic of high severity has been observed in Dangyang County, Hubei province, China. Initial symptoms consisted of multiple, small, irregular to oval, white leaf spots, which enlarge to produce sunken purple lesions, sometimes surrounded by a bright yellow margin. As the disease progressed, lesions expanded and merged, resulting in withering of leaf tips. After isolation and pathogenicity testing, the causal agent of leaf blight of garlic was identified as Stemphylium solani from cultural and morphological characteristics, and subsequent analysis of the internal transcribed spacer region of ribosomal DNA. When fungal plugs of two S. solani isolates were inoculated onto 11 garlic cultivars and 20 other crop species, leaf spots appeared on all inoculated plants, but two garlic cultivars (Qingganruanye and Ruanruanye) and three crop species (Capsicum annuum, Brassica napus and Amaranthus mangostanus) showed the smallest leaf spots. In cross-inoculation experiments, no indications of host specificity were observed, but S. solani isolated from garlic was generally the most virulent on five plant species, while the isolate from leek (Allium odorum) was generally the least virulent. Toxicity testing of the crude culture filtrates indicated that garlic isolates produced toxin(s) that were not heat-labile and induced different levels of phytotoxicity toward various garlic cultivars and crops.     

Epidemiology of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">syringae</Emphasis> on tomato

Leaf spot of tomato, incited by Pseudomonas syringae pv. syringae, has been reported recently in Italy on grafted and non-grafted tomato plants (scion Cuore di Bue, rootstock Solanum lycopersicum x Solanum hirsutum cv. Beaufort). In some greenhouses, more than 80% of plants were affected, with a marked reduction in yield. This work was undertaken in order to understand the effect of the number of hours of incubation at high relative humidity (r.h.) and temperature as well as the effect of the presence of wounds at infection time on the development of leaf spot. A difference in sensitivity to leaf spot was observed in the various cultivars tested, in terms of severity of P. syringae pv. syringae, with “Cuore di Bue” being the most susceptible of these cultivars. The development of leaf spot is mostly favored by the presence of wounds, at temperatures between 15 and 20°C. The severity of the disease is lower at 10 and 25°C and very low at 30°C. Under the most favorable temperature conditions, the presence of wounds is sufficient to allow the development of the pathogen immediately upon incubation at high r.h. The effect of wounds and the relatively low requirement of hours of incubation at high r.h. suggest the need for careful management and handling of plants when temperatures range between 15 and 25°C, and particularly within 15 and 20°C. All operations carried out, particularly at transplant and immediately after, should avoid the creation of wounds.     

Response of UK winter wheat cultivars to <Emphasis Type="Italic">Soil-borne cereal mosaic</Emphasis> and <Emphasis Type="Italic">Wheat spindle streak mosaic viruses</Emphasis> across Europe.

Twenty-one UK winter wheat cultivars were grown over three seasons at sites with natural inoculum sources of Soil-borne cereal mosaic virus (SBCMV) and Wheat spindle streak mosaic virus (WSSMV) located in France, Italy and the UK. Plants were assessed visually for virus symptoms and leaf extracts were tested for the presence of each virus using enzyme-linked immunosorbent assays (ELISA). Cultivars showing little or no foliar symptoms and low levels of virus in leaf tissue were classified as resistant to each virus. All the trials were taken to harvest and agronomic data collected. At the most heavily infected sites, severe symptoms of SBCMV were observed in all UK cultivars except Aardvark, Charger, Claire, Cockpit, Hereward and Xi 19. The latter cultivars exhibited either light or no symptoms and little or no SBCMV infection in leaves. In fields with WSSMV, the virus failed to develop in Italy, but was detected in the leaves of all the susceptible control cultivars at a site in France. However, no UK cultivar tested positive for WSSMV. Multi-site analysis indicated that the presence of WSSMV did not increase the impact of SBCMV on the height, thousand-grain weight or yield of UK cultivars. The wheat cultivars on test gave a similar response to SBCMV across three European countries. Possible sources of SBCMV resistance are discussed.     

Potential inhibitors against <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>, produced by the fungus <Emphasis Type="Italic">Myrothecium</Emphasis> sp. associated with the marine sponge <Emphasis Type="Italic">Axinella</Emphasis> sp.

Sclerotinia sclerotiorum is a worldwide ascomycete fungal plant pathogen, which causes enormous yield losses on major economic crops such as crucifers, grain legumes and several other plant families. The objective of this research was to isolate and characterise some bioactive products from cultures of fungi associated with the marine sponge Axinella sp. In total, nine fungal isolates were obtained from the marine sponge Axinella sp. collected from the South China Sea. A group of test strains, including two G⁺ strains (Bacillus subtilis and Staphylococcus aureus), two G⁻ strains (Escherichia coli and Pseudomonas aeruginosa) and three fungi including two plant pathogenic fungi Sclerotinia sclerotiorum and Magnaporthe grisea and Saccharomyces cerevisiae, were employed as the indicator organisms for bioactivity screening. Using antagonistic tests and bioactive screening of the ethyl acetate (EtOAc) extracts of the corresponding cultures, fungal isolate JS9 showed the stronger efficacy against the test indicator strains, especially the indicator fungal pathogens. Isolate JS9 was further identified as Myrothecium sp. by a combination of morphological features and 18S rDNA BLAST on GenBank. Two macrocyclic trichothecenes, roridin A (compound 1) and roridin D (compound 2) were purified by tracking the activity of the EtOAc extract fractions and characterised with spectral analyses including MS, ¹H-NMR, ¹³C-NMR and disortionless enhancement by polarization transfer (DEPT). In vitro antifungal tests showed that the two macrocyclic trichothecenes were bioactive against S. cerevisiae, M. grisea and S. sclerotiorum with minimal inhibitory concentrations of 31.25, 125 and 31.25 μg ml⁻¹ for roridin A, and 62.5, 250 and 31.25 μg ml⁻¹ for roridin D, respectively. The present investigation demonstrated that two antifungal trichothecenes including roridin A and roridin D produced by the fungus Myrothecium sp. isolated from the marine sponge Axinella sp. could be potential inhibitors against the plant pathogen S. sclerotiorum. Lian Wu Xie and Shu Mei Jiang contributed equally to this work.     

Fusarium rot of hyacinth caused by <Emphasis Type="Italic">Gibberella zeae</Emphasis> (anamorph: <Emphasis Type="Italic">Fusarium</Emphasis><Emphasis Type="Italic"> graminearum</Emphasis>)

Severe rot of leaves, peduncles and flowers caused by Gibberella zeae (anamorph: Fusarium graminearum) was found on potted plants of hyacinth (Hyacinthus orientalis), a liliaceous ornamental, in greenhouses in Kagawa Prefecture, Japan, in January 2001. This disease was named “Fusarium rot of hyacinth” as a new disease because only the anamorph, F. graminearum, was identified on the diseased host plant. The authors contributed equally to this work. The fungal isolate and its nucleotide sequence data obtained in this study were deposited in the Genebank, National Institute of Agrobiological Sciences and the DDBJ/EMBL/GenBank databases under the accession numbers MAFF239499 and AB366161, respectively.     

Genetic variation among <Emphasis Type="Italic">Fusarium</Emphasis> isolates from onion,and resistance to <Emphasis Type="Italic">Fusarium</Emphasis> basal rot in related <Emphasis Type="Italic">Allium</Emphasis> species

The aim of this research was to study levels of resistance to Fusarium basal rot in onion cultivars and related Allium species, by using genetically different Fusarium isolates. In order to select genetically different isolates for disease testing, a collection of 61 Fusarium isolates, 43 of them from onion (Allium cepa), was analysed using amplified fragment length polymorphism (AFLP) markers. Onion isolates were collected in The Netherlands (15 isolates) and Uruguay (9 isolates), and received from other countries and fungal collections (19 isolates). From these isolates, 29 were identified as F. oxysporum, 10 as F. proliferatum, whereas the remaining four isolates belonged to F. avenaceum and F. culmorum. The taxonomic status of the species was confirmed by morphological examination, by DNA sequencing of the elongation factor 1-α gene, and by the use of species-specific primers for Fusarium oxysporum, F. proliferatum, and F. culmorum. Within F. oxysporum, isolates clustered in two clades suggesting different origins of F. oxysporum forms pathogenic to onion. These clades were present in each sampled region. Onion and six related Allium species were screened for resistance to Fusarium basal rot using one F. oxysporum isolate from each clade, and one F. proliferatum isolate. High levels of resistance to each isolate were found in Allium fistulosum and A. schoenoprasum accessions, whereas A. pskemense, A. roylei and A. galanthum showed intermediate levels of resistance. Among five A. cepa cultivars, ‘Rossa Savonese’ was also intermediately resistant. Regarding the current feasibility for introgression, A. fistulosum, A. roylei and A. galanthum were identified as potential sources for the transfer of resistance to Fusarium into onion.     

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.     

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.     

Cytology of infection of <Emphasis Type="Italic">Fusarium mangiferae</Emphasis> Britz in different malformed reproductive parts of mango

Aetiology of mango malformation (MM) has intrigued the scientists since its inception. The objective of the study was to glean an insight into association of the fungus, Fusarium mangiferae, with different malformed regions, viz. panicle-shoot juncture, apical buds, primary and secondary peduncles, in five exotic mango cultivars. Tissue assays revealed an infection of 88.5, 84.75 and 82.5% in cvs Zill, Sensation and Tommy Atkins, respectively. Least infection of 69.75% was found in cv. Keitt. No exotic cultivar was found to be free of fungal infection. Apical buds proved to be the potential infection site of the fungus amongst the four malformed regions, hosting maximum within-tissue infection of 86.2%. Determination of F. mangiferae at proximal and distant sites of the malformed panicles exhibited maximum recovery of 82.0% at 0 cm and only 3% at >30 cm distance beneath the panicle. In the case of non-malformed panicles, an infection of 14.0% was recorded at 0 cm distance while no detection could be made from non-malformed branches. Moreover, examination of ultra-thin bud sections under Transmission Electron Microscopy (TEM) revealed inter- and intra-cellular ramification of fungal hyphae, indicating fungal ingress in malformed bud tissues of local cv. Malda. The present studies explored the sites hosting the causal fungus in mango and provide convincing evidence that F. mangiferae is responsible for turning healthy tissues into the malformed condition. These findings suggest that inoculum specific management strategies are needed in future to curb malformation disease in mango orchards.     

BABA effects on the behaviour of potato cultivars infected by <Emphasis Type="Italic">Phytophthora infestans</Emphasis> and <Emphasis Type="Italic">Fusarium solani</Emphasis>

Since most plants possess resistance mechanisms which can be induced upon pre-treatment with a variety of chemical compounds, the use of β-aminobutyric acid (BABA) as a defence inducer without reported toxic effect on the environment was studied. The aim of this work was to analyse the effectiveness of BABA to induce resistance against Phytophthora infestans and Fusarium solani in potato cultivars differing in their level of resistance to late blight. The behaviour of some components of biochemical mechanisms by which BABA increases resistance against P. infestans, as well as the effect of BABA on the activity of a potential pathogenic factor of F. solani, were studied. Plants with four applications of BABA throughout the crop cycle produced tubers more resistant to P. infestans and F. solani than non-treated plants. In addition, tuber slices from treated plants, inoculated with P. infestans, showed an increase in phenol and phytoalexin content. The aspartyl protease StAP1 accumulation was also higher in tubers obtained from treated plants and inoculated with P. infestans. This result was observed only in the more resistant potato cv. Pampeana, early after infection. In the potato–F. solani interaction, infected tubers coming from BABA-treated plants showed minor fungal proteolytic activity than infected, non-treated ones. For potato cvs Pampeana and Bintje, the BABA treatment improved the yield of harvested tubers. The number of tubers per plant and total weight of harvested tubers was greater for those obtained from treated plants with two early or four applications of BABA. The results show that the BABA treatment increases the resistance of potatoes but the degree of increase depends on the original level of resistance present in each cultivar.     

Cytological events in tomato leaves inoculated with conidia of <Emphasis Type="Italic">Blumeria graminis</Emphasis> f. sp. <Emphasis Type="Italic">hordei</Emphasis> and <Emphasis Type="Italic">Oidium neolycopersici</Emphasis> KTP-01

Leaves of tomato and barley were inoculated with conidia of Blumeria graminis f. sp. hordei race 1 (R1) or Oidium neolycopersici (KTP-01) to observe cytological responses in search of resistance to powdery mildew. Both conidia formed appressoria at similar rates on tomato or barley leaves, indicating that no resistance was expressed during the prepenetration stage of these fungi. On R1-inoculated tomato leaves, appressoria penetrated the papillae, but subsequent haustorium formation was inhibited by hypersensitive necrosis in the invaded epidermal cells. On the other hand, KTP-01 (pathogenic to tomato leaves) successfully developed functional haustoria in epidermal cells to elongate secondary hyphae, although the hyphal elongation from some conidia was later suppressed by delayed hypersensitive necrosis in some haustorium-harboring epidermal cells. Thus, the present study indicated that the resistance of tomato to powdery mildew fungi was associated with a hypersensitive response in invaded epidermal cells but not the prevention of fungal penetration through host papilla.     

Reaction of <Emphasis Type="Italic">Leersia</Emphasis> grasses to <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> collected from Japan and Asian countries

The present study was conducted to determine if there is specificity in the host-pathogen relationship between the isolates of Xanthomonas oryzae pv. oryzae, the causal bacterium for rice blight and Leersia grasses, the alternative weed hosts of the disease. Plants of three species of Leersia, namely, L. sayanuka, L. oryzoides and L. japonica, were collected from various parts of Japan and were inoculated with the X. oryzae pv. oryzae isolates obtained from various locations in Japan and from 11 Asian countries. Four L. sayanuka plants were found susceptible to all Race II isolates and some Race I isolates, but were resistant to all Race III isolates. Race III is known to have a wider range pathogenicity to rice cultivar groups compared with Race I and II. Although the reactions of two L. oryzoides plants to Race I and II isolates were similar to that of L. sayanuka, the L. oryzoides plant collected from Niigata Prefecture showed a susceptible reaction to some Race III isolates. On the other hand, L. japonica plants gave reactions different those of L. sayanuka and L. oryzoides, with two plants of L. japonica found to be resistant to all test isolates collected from Japan. The Asian isolates exhibited a wide host range against the international differential rice cultivars, but almost all of them were avirulent to Leersia plants. These results indicate that the relationship between the pathogenicity of the causal bacterium and the resistance of host plants is very complex, and suggest that pathogenic diversity of X. oryzae pv. oryzae might be related to the resistance of Leersia spp.