 |
||||||||||||||||||||||||||||||||
|
 |
Journal of Plant Pathology (2009), 91 (2) INVITED REVIEW Viroids are autonomously replicating, small singlestranded
circular RNA molecules that do not code for
proteins and may cause disease in infected, susceptible
plants. Viroids have the ability to induce both RNA-mediated
transcriptional gene silencing (TGS) and posttranscriptional LETTER TO THE EDITOR PCR-based methods offer advantages over more traditional diagnostic tests, in that organisms do not need to be cultured prior to their detection and protocols are highly sensitive and rapid. Consequently, there is a shift in research towards DNA-based techniques. Although reports already exist on a variety of PCR-based fingerprinting assays used to analyse the genetic diversity of bacterial populations and define their relationships, this review focuses on the general use of PCR in phytobacteriology for detection and diagnosis purposes. An updated and detailed list of published PCR protocols for detection and identification of plant-pathogenic bacteria is presented and discussed, aimed at facilitating access to information that could be particularly useful for diagnostic laboratories. This compilation includes and discusses 246 articles published between 1989 and 2007 addressing 23 genera, more than 50 species, 10 subspecies and more than 40 pathovars. VIABILITY OF PYRENOPHORA GRAMINEA CULTURES AFTER SUNLIGHT EXPOSURE UNDER FIELD CONDITIONS Conidia of Pyrenophora graminea, the causal agent of barley leaf stripe, were simultaneously exposed outdoors to direct solar radiation or placed in an adjacent ventilated enclosure in the dark for four months. After exposure, conidia were placed on water agar in closed Petri dishes and allowed to germinate for 24 h. Shaded conditions were always more favourable to conidia germination and mycelial growth than sunlight conditions. Significant decreases (P<0.05) in conidia germinability (60.6%) and mycelial growth (41.46%) were detected in light-exposed conidia in comparison with the non-exposed control. Exposure also decreased the pathogenicity of fungus on different cultivars. The possibility that sunlight exposure may reduce conidia germinability over the hottest four months and aerial transport distances should be considered. OCCURRENCE, DISTRIBUTION, CHARACTERIZATION
OF CITRUS TRISTEZA VIRUS AND ITS VECTORS IN SYRIA Syrian citrus growing areas (Lattakia and Tartous) were surveyed to assess the distribution of Citrus tristeza virus (CTV) and its vectors. Eight nurseries, two budwood source fields and 19 groves of the main citrus varieties were visually inspected and sampled for laboratory analysis. A total of 89 CTV-infected plants out of 2653 were identified by DTBIA in two nurseries, two budwood source fields and six groves. Based on the reaction with several monoclonal antibodies (MAbs), CTV isolates clustered in 4 serogroups. Only one CTV strain from the sweet orange of cv. Valencia induced severe symptoms on Mexican lime seedlings by grafttransmissions. Hybridization of PCR amplicons from the coat protein gene with a set of strain-specific probes revealed a complex strain mixture. Aphis spiraecola (50%) and A. gossypii (27%) were the most common aphids, whereas no evidence for the presence of Toxoptera citricidus was found. SPECIFIC DIGOXIGENIN-LABELLED RIBOPROBES FOR DETECTION A dot-blot hybridization protocol using digoxigeninlabelled riboprobes was finalized for the detection of Citrus psorosis virus (CPsV) and Citrus variegation virus (CVV). Both viruses were readily identified in different organs of screenhouse-grown and, throughout a 9- month period, in-field-grown citrus plants. With CPsV, strong hybridization signals were obtained by dot blot hybridization from flowers (ovaries) and young leaves and, with CVV, from young leaves and shoots. In tissues prints, CPsV was satisfactorily detected from ovaries and CVV from petioles, ovaries, young leaves and tender shoots. The sensitivity threshold of the assay was determined to be 75 pg of in vitro transcripts for both CPsV and CVV, allowing detection of both viruses even when their titres decreased in plant tissues and ELISA tests failed. DIAGNOSIS OF EUTYPA LATA INFECTION IN GRAPEVINES BY SEROLOGICAL DETECTION
OF SECRETED POLYPEPTIDES Eutypa lata is the causal agent of a devastating disease affecting grapevines around the world. We have previously reported that the fungus secretes in its culture medium a variety of polypeptides, and we have developed serological method of detecting these. Rabbit antibodies raised against the polypeptide fraction, recognized secreted fungal proteins with high sensitivity (commonly 1 ng). These antibodies were specific since they cross-reacted with polypeptides secreted by various strains of Eutypa lata but not by other fungal pathogens involved in other wood decay diseases, namely esca (Phaeomoniella chlamydospora and Phaeoacremonium aleophilum) and black dead arm (Diplodia seriata and Neofusicoccum parvum). These results were obtained by ELISA assay and immunolocalization on ultrathin sections. The ability of the polypeptides to be transported in vines permitted development of a reliable dot-blot method of diagnosis rapid, easy to perform and not destructive for grapevines. Advantages and drawbacks of the method are discussed. COMPETITION FOR CELLULOSE EXPLOITATION BETWEEN RHIZOCTONIA SOLANI AND TWO TRICHODERMA ISOLATES IN THE DECOMPOSITION OF WHEAT STRAW The competitive saprophytic ability (CSA), expressed as competition for wheat straw, between Rhizoctonia solani and two antagonistic strains of Trichoderma (T. virens I10 and T. asperellum I252) was evaluated. Both antagonists competed with R. solani for wheat straw possession for up to 10 days. Later, straw baits were occupied by R. solani, but the rates depended on the antagonist present. Cellulolytic activities were evaluated as a possible mechanism for maintenance of the resource, since the major components of wheat straw are cellulose and hemicelluloses. Mycelial protein content was used to estimate fungal growth, with sucrose or straw as the only carbon source. R. solani grew equally well with sucrose or straw, whereas the antagonists did better on straw. Levels of extracellular proteins produced by the three isolates changed with the carbon source, being highest in straw for all fungi tested. Among the enzymatic activities tested, only cellulase activity of T. virens I10 decreased with time and this correlated with reduction in competitive ability. We conclude that efficient exploitation of cellulose plays a role in competition for straw. ANTIMICROBIAL ACTIVITIES OF LEAF EXTRACTS OF PISTACIA Leaf extracts of Pistacia vera, Pistacia atlantica, Schinus terebenthifolius and Schinus molle were investigated for their antimicrobial effect against Agrobacterium tumefaciens, Pseudomonas savastanoi pv. savastanoi, Fusarium solani and Rhizoctonia solani. Extracts were prepared from dried and powdered leaves with solvents (hexane, ethanol, methanol and water) with different degrees of polarity. Aqueous and methanolic extracts exhibited a high level of antifungal and antibacterial activity. Leaf extracts from P. atlantica showed a high level of antibacterial activity. However, leaf extracts from Schinus terebenthifolius and Schinus molle showed the best antifungal activities. Total polyphenol contents of extracts was positively correlated with the diameter of the inhibition zones suggesting their potential antimicrobial activity. A preliminary phytochemical screening revealed the presence of tannins, flavanoids and alcaloids. These findings suggest that leaves of Pistacia and Schinus spp. are potential sources of antimicrobial compounds. EFFECTS OF BASIDIOMYCETE LACCASE ON CERCOSPORIN Cercosporin is a perylenequinone pigment produced by fungi in the genus Cercospora which under light generates reactive oxygen species causing membrane damage and mortality of living cells. Our objectives were to demonstrate that fungal laccase, a lignolytic copper-containing enzyme, can degrade cercosporin and reduce cercosporin toxicity toward living cells. Cercosporin from Cercospora beticola and Cercospora hayi was treated with laccase from basidiomycete fungi (Pleurotus ostreatus and Trametes versicolor) in the dark and under constant light. Under these conditions the absorbance of the cercosporin decreased at 220, 279 and 295-500 nm within 10 min of reaction with laccase from either P. ostreatus or T. versicolor, indicating that basidiomycete laccase can induce changes in UV-visible spectra of cercosporin. The LIVE/DEAD® Bac LightTM VIABILITY kit and fluorescent microscopy showed more viable E. coli cells after incubation under light with cercosporin and laccase than with cercosporin alone. Lesions were apparent on sugar beet leaves exposed to cercosporin under light after 48 h, but leaves exposed to cercosporin and laccase showed visibly less damage. These data suggest that laccase from basidiomycete fungi can decrease the toxic effect of cercosporin toward microorganisms and plant tissue. EFFECTS OF CITRUS TRISTEZA VIRUS ON THE GROWTH
OF IN VITRO-CULTURED CITRUS Citrus tristeza virus isolate YC (CTV-YC), a stem pitting- inducing strain, was graft-transmitted to seedlings of Mexican lime [Citrus aurantifolia (Christm.) Swingle], Pineapple sweet orange [C. sinensis (L.) Osbeck] and Arizona Etrog citron 861-SI (C. medica L.). Nodal stem segments from CTV-YC-infected and healthy seedlings of the three species were used as explants for regeneration in vitro. CTV presence reduced significantly the proliferation rate, the vigour and the rooting capacity of regenerated shoots. The most severe effect was observed on Etrog citron, whose regeneration ratio, shoot proliferation and root formation were inhibited to a great extent. CTV infection affected also the survival of transplanted plantlets. In vitro shoot cultures from CTV-infected explants of sweet orange and Mexican lime were successfully sub-cultured for up to 20-24 months, whereas those from infected Etrog usually survived less than 7 months. CTV could be detected in all sub-cultures, but its titer decreased with time. DETECTION OF PSEUDOMONAS AVELLANAE AND THE BACTERIAL MICROFLORA
OF HAZELNUT AFFECTED BY ‘MORIA’ IN CENTRAL ITALY The presence of Pseudomonas avellanae and the bacterial flora associated with different hazelnut organs, was monitored from 2004-2007 in two areas of the province of Viterbo (central Italy). Samples were randomly selected from three orchards affected by the disease known as ‘Moria’ (dieback): symptomatic twigs or branches (2004-2005 and 2007), symptomless suckers (2004-2006), leaves and pollen (2006-2007). The presence of P. avellanae was examined by PCR assay (PCRWA/WC) and also checked by isolation, enabling us to verify the reliability of the PCR. The incidence of P. avellanae in symptomatic samples ranged from 9 to 38% in the areas monitored. P. avellanae was found in up to 3% of symptomless suckers whereas pollen and leaves all tested negative. PCRWA/WC proved to be more reliable than isolation for the detection of P. avellanae. The association of bacteria with different hazelnut organs was assessed by isolation followed by 16S rDNA sequencing. The recovered isolates were closely related to bacteria mainly associated with the environment, with the exceptions of Brenneria quercina and Pseudomonas syringae. B. quercina was rarely recovered, and only from leaf surfaces. On the other hand, P. syringae pv. syringae was frequently isolated from buds, bark tissue and leaves. A preliminary characterization of this P. syringae population by rep-PCR is reported. CONTROL OF POWDERY MILDEW ON CUCUMBER COTYLEDONS BY CHITOSAN The objective of this study was to determine the effects of chitosan to control powdery mildew on cucumber cotyledons inoculated with races 1, 2 and 5 of Sphaerotheca fuliginea and race 1 of Erysiphe cichoracearum. Chitosan, an elicitor of plant defences and also an inhibitor of fungal growth, was sprayed on the upper surface of cotyledons at concentrations 1% and 2.5%. Control cotyledons were sprayed with sterile water only. When cotyledons had dried they were inoculated by applying eight 5 μl droplets of a conidial suspension (4×104 or 4×105 spores ml-1) on the adaxial surface of each cotyledon. The number of lesions developed (mildew incidence) and lesion size were evaluated ten days after inoculation. The highest chitosan concentration significantly inhibited the mildew incidence on pretreated cotyledons inoculated with races of S. fuliginea and race 1 of E. cichoracearum, at both conidial concentrations compared to control. Significant differences in lesion diameters were observed between chitosan-treated cotyledons and controls after inoculation with S. fuliginea and E. cichoracearum when 4×105 spores ml-1 were used. SYMPTOMS AND FUNGI ASSOCIATED A field survey was carried out in Catalonia (northeast Spain) to characterize the decline of mature grapevines. The relationships of both external and internal symptoms of diseased plants and their associated mycoflora were studied. Co-occurrence of different internal disease symptoms was frequently observed, since 44% of the sampled plants had wood lesions commonly associated with at least two of the following decline diseases: eutypiose, black dead arm or esca. The results obtained suggest that apoplexy might not be associated only with esca-affected plants, since 60% of surveyed vines showing apoplexy showed also V-shaped necroses, which are commonly associated with eutypiose and black dead arm, and 20% were exclusively affected by V-shaped necroses. An experiment was conducted to establish the pathogenicity of the most representative fungi isolated from diseased tissues of declining plants, by artificially inoculating 1-year-old vines of cvs Macabeo and Tempranillo. As indicated by the extension of the vascular lesions, pathogenicity was confirmed for most of the species tested, namely Botryosphaeria dothidea, Diplodia seriata, Eutypa lata, Neofusicoccum luteum, N. parvum and Phaeomoniella chlamydospora. ALLELE-SPECIFIC REAL-TIME PCR FOR QUANTIFICATION AND DISCRIMINATION
OF STEROL 14 α-DEMETHYLATION-INHIBITOR-RESISTANT GENOTYPES OF MYCOSPHAERELLA GRAMINICOLA The level of resistance of Mycosphaerella graminicola to sterol 14 α-demethylation inhibitors (DMIs) is characterised by point and deletion mutations in the CYP51 gene that encodes sterol 14 α-demethylase. Rapid and pre-symptomatic detection of these mutations is required for effective control by fungicides. In this study, an allele-specific real-time PCR method was developed. An additional mismatched nucleotide at the third position from the single nucleotide polymorphism at the 3- prime end of each allele-specific primer stops non-specific PCR amplification. Minor groove binding specific probes were designed to quantify general strains of M. graminicola and strains that contain isoleucine at position 381 of the CYP51 protein sequence. Specific amplification of the target alleles was reproducible. A high level of discrimination between genotypes using pure fungal DNA was confirmed in vivo, based on leaf samples collected from different wheat growing regions in France. A high level of I381V-genotypes (>70%) was found in all samples. The results showed that allele-specific real-time PCR allows pre-symptomatic and accurate quantitative detection of DMI-resistant genotypes of M. graminicola, with a shorter turnaround time compared to conventional methods. The simplicity and effectiveness provided by intentional mismatch primers offer a broad range of applications for laboratory and field analysis. THE SENSITIVITY OF ASPERGILLUS NIGER
AND FUSARIUM OXYSPORUM f. sp. CEPAE TO FUNGISTASIS IN ONION-GROWING SOILS Twenty-seven soil samples were collected from the onion (Allium cepa L.) fields of Tekirdag province, Turkey. These samples were investigated for the sensitivity of Aspergillus niger V. Tieghem (AN) and Fusarium oxysporum Schlechtend.: Fr. f. sp. cepae (H. N. Hans.) W. C. Snyder H. N. Hans (FOC), known as the causal agents of black mould and basal rot of onion, respectively, to soil fungistasis. Fungistasis was evaluated using two methods: inhibition of pathogen spore germination by volatile compounds from the soil and determination of the antagonistic fungal population of soil samples. Volatile compounds in twelve of the soil samples strongly (>70%) inhibited spore germination of only AN. Inhibition rates of volatile compounds were not correlated with physical and chemical characters of the soils. Fungi isolated from soil samples were evaluated for their antagonism to both pathogens using dual cultures. The population of species causing over 70% inhibition of radial growth on pathogens was calculated in soil samples. The presence of both volatile compounds inhibiting spore germination of AN and populations of fungi antagonistic for AN and FOC were observed in six of the soil samples. The possible effects of two fungistatic mechanisms in soils on disease development by these pathogens are discussed. GENETIC VARIABILITY OF STOLBUR PHYTOPLASMA IN ANNUAL CROP AND WILD PLANT SPECIES
IN SOUTH MORAVIA The genetic diversity of stolbur phytoplasma isolates was assessed by different molecular typing methods in a wide range of plant hosts in a limited geographical area of the Czech Republic. Plants of Apium graveolens, Capsicum annuum, Solanum lycopersicum, Solanum tuberosum, Amaranthus retroflexus, Calystegia sepium, Cirsium arvense, Convolvulus arvensis, and Datura stramonium displaying symptoms of proliferation, discoloration, and malformation of flower were collected in vegetable crop plots in the vicinity of the village of Lednice (south Moravia) and examined for the presence of phytoplasmas. Using 16S rDNA amplification and RFLP assays, stolbur phytoplasma was detected in some of the symptomatic plants. The genotype of the phytoplasma isolates detected was investigated by restriction pattern or sequence analysis of two non-ribosomal house-keeping genes (tuf and secY) and a recently characterized polymorphic gene (vmp1). According to tuf genotyping, all isolates were of the tufAY-b genotype sensu Langer and Maixner (2004), a genotype known to be associated with bindweed (C. arvensis) reservoirs. A finer isolate differentiation was obtained with vmp1 genotyping as four genetic variants carrying different vmp1 RFLP patterns were found, especially in C. arvensis. In addition, preliminary sampling in other south Moravian localities and secY sequence analyses diclosed also the presence of stolbur isolates different from those previously reported in binweed (C. arvensis) and grapevine. USING TERMINAL RESTRICTION FRAGMENT LENGTH POLYMORPHISM (T-RFLP) TO MONITOR CHANGES IN FUNGAL POPULATIONS
ASSOCIATED WITH PLANTS Terminal restriction fragment length polymorphism (T-RFLP) has been widely used as a method for analysing changes in microbial populations. Here, the technique has been further developed using Gaeumannomyces graminis var. tritici (Ggt) as a model system, to show that it can be used as a semi-quantitative measurement of fungal pathogens associated with the roots of plants, and to monitor the survival of pathogens in the soil. To provide an internal reference for measuring fluxes in Ggt populations, primers were designed to amplify products from the fungal ITS2 region and simultaneously from wheat root DNA. Peak height ratios for the fungus relative to the wheat control were then calculated and these ratios were then compared between samples to measure changes in populations. The results showed that the ratio of the Ggt TRF peak height to the wheat TRF peak height could be used as a measure of the relative amount of Ggt in a sample. These ratios correlated well with disease index scores and also with fungal biomass as assessed by determination of ergosterol content using HPLC. T-RFLP can therefore be used directly as a tool for comparison of the relative levels of Ggt and other fungi on different root samples, and by incorporating fixed amounts of wheat DNA as an internal standard into soil samples prior to DNA extraction, the same method could be used to measure fluxes in soil populations of fungi. DETECTION OF FLMaV-1 AND FLMaV-2 IN THE MEDITERRANEAN REGION AND STUDY ON SEQUENCE VARIATION OF THE HSP70 GENE The presence of Fig leaf mottle-associated virus 1 (FLMaV-1) and Fig leaf mottle-associated virus 2 (FLMaV-2) was investigated in fig orchards of six Mediterranean countries. A total of 415 samples coming from Albania, Algeria, Lebanon, Syria, Tunisia and Italy were collected and tested by PCR, which detected the presence of at least one of the tested viruses in all surveyed countries. Except for Algeria and Syria, where FLMaV-1 was absent, both viruses were found in all surveyed countries with mean infection rates of 34% and 18.5% for FLMaV-1 and FLMaV-2, respectively. More than 7% of the samples were infected with both viruses. As ascertained by dsRNA analysis, 25 out of 65 samples (ca. 38.5%) showed visible bands in polyacrylamide gel electrophoresis. Single strand conformation pattern (SSCP) analysis of a portion of the viral HSP70 gene from figs of different geographical origin showed heterogeneous patterns for both viruses. The HSP70 gene variability was confirmed by sequence analysis. The level of nucleotide variability between the isolates reached 18% for FLMaV-1 and 15% for FLMaV-2. While the deduced amino acid sequence variability maintained almost the same range of divergence in the case of FLMaV-1 (19%), this level decreased to 6% in the case of FLMaV- 2. The position of the isolates in phylogenetic trees constructed with HSP70 sequences did not correspond to their geographical origin, except for the Lebanese isolates which grouped together in a single cluster. DETECTION OF XANTHOMONAS AXONOPODIS pv. VESICATORIA Bacterial spot, caused by Xanthomonas axonopodis pv. vesicatoria, is one of the most important diseases of pepper in Turkey, where the disease has been epidemic in the last six years in the eastern Mediterranean region. The incidence and importance of natural infection in pepper seeds produced in the region was investigated in the present study. A total of 29 seed samples were tested by immunofluorescence assay using a commercial specific antibody, semi-selective isolation on medium Tween B, and a seedling screening tests, where plantlets were examined for typical spot symptoms on cotyledons 7-14 days after emergence. Pathogen populations were found to be in the range of 5×101-5×104 cells/g seed when using semi-selective medium Tween B. The diseased seeds as determined in the seedling screening ranged from 7 to 36%; these figures agreed with numbers found in the Tween B medium test. The strains isolated on Tween B were identified by PCR using primer designed on the hrp gene specific for Xanthomonas spp., and pathogenicity on pepper plants. Twenty-one out of 29 seed samples were found to be contaminated by Xanthomonas axonopodis pv. vesicatoria. On the basis of these findings, infested seeds must be seen as a major source of inoculum for this disease in the region. CONTROL OF PENICILLIUM DIGITATUM ON ORANGE FRUIT The antagonistic bacterium, Pantoea agglomerans HR, was evaluated for controlling citrus green mould caused by Penicillium digitatum Sacc. at 20°C (room temperature) and 4°C (cold storage). This isolate was also assessed in combination with dipping in 3% sodium bicarbonate solution at 24°C and 45°C on artificially inoculated Thomson navel oranges. Application of the antagonist alone reduced green mould by more than 75% at both temperatures, but was not as effective as Imazalil (more than 87% decay reduction). The antagonistic bacterium was completely tolerant to sodium bicarbonate up to a concentration of 3%. In addition, its efficacy for controlling green mould was improved at least by 5% and 11% when combined with 3% sodium bicarbonate at 24°C and 45°C, respectively. CHARACTERISING THE PATHOGENICITY DIVERSITY Fifty nine isolates of Ustilaginoidea virens, the cause of false smut of rice (Oryza sativa L.), were obtained from 46 rice hybrids in 14 counties in Sichuan, China during a survey conducted in 2006. Their pathogenicity was tested by inoculation on 3 rice hybrids, Gangyou182, Gangyou94- 11 and Yixiangyou2292, 6-9 days before heading in 2007. False smut was assessed 3 weeks after inoculation using a disease index (DI) based on symptom frequency on the panicles. The sporulation capacity of these isolates was also measured. The results showed that: (i) DIs were significantly different (P<0.01) both among pathogen isolates and rice hybrids, ranging from 0 to 98.52; (ii) there was a significant interaction between isolates and hybrids (P<0.01); (iii) significant differences in sporulation among the 59 isolates were found (P<0.01), but no relationship between sporulation and virulence on the 3 hybrids; (iv) variation in sporulation was observed among isolates originating from different counties and from the same county (P<0.05); (v) there were significant differences (P<0.01) between isolate groups from different host origin, female parents and male parents. Our results indicated a linkage between the pathogenicity of U. virens isolates and the resistance of rice hybrids. The 59 isolates could be classified into 6 groups based on their virulence to the tested rice hybrids. While variation in sporulation did not indicate host genotype-pathogen isolate interaction, pathogenicity data suggest specialisation, which is dependent on the site of origin of the isolates, the original host (rice hybrids) and the parentage of the original host. MOLECULAR CHARACTERIZATION AND TRANSMISSION Safflower plants with phyllody symptoms were observed in Fars and Yazd provinces of Iran. Affected plants showed floral virescence, phyllody and proliferation, proliferation of axillary buds along the stem and little leaf symptoms. The causal agent (SP) was transmitted from diseased to healthy safflower by grafting and from safflower to safflower and periwinkle by dodder (Cuscuta campestris). The presence of phytoplasmas in diseased plants was shown by direct and nested polymerase chain reaction assays using phytoplasma-specific primer pairs P1/P7 and R16F2n/R2. Restriction fragment length polymorphism (RFLP) analysis of nested R16F2n/R2 primed PCR product (1.2 kb) classified SP phytoplasma in the clover proliferation phytoplasma group (16SrVI). Sequence homology, phylogenetic and putative restriction site analyses of 16S rRNA gene also identified SP phytoplasma as a member of 16SrVI group. On the basis of molecular analyses, SP phytoplasma was most closely related to brinjal little leaf and periwinkle little leaf phytoplasmas, members of subgroup 16SrVI-C. SHORT COMMUNICATION Maize ear rot is an important disease of maize in Kenya. In this study fumonisin B1 (FB1) and aflatoxin B1 (AFB1) analysis were conducted on symptomless and rotten maize harvested at different harvest time points after physiological maturity (HTPAPM). Fusarium verticillioides dominated at all HTPAPM. Other fungi reported include Aspergillus flavus, Aspergillus parasiticus and Sternocarpella maydis. In 2001, FB1 levels in symptomless maize ranged between 22 to 1348 μg/kg. Mean FB1 levels at 4, 8, and 12 weeks HTPAPM for Malava were 56, 80 and 317 μg/kg respectively. In Tongaren during 2001 mean FB1 levels of 41, 179 and 590 μg/kg were recorded at 4, 8, 12 week HTPAPM respectively. The FB1 levels in rotten maize ranged from 39 to >5000 μg/kg and increased with HTPAPM. The highest AFB1 level was 17 μg/kg in rotten maize. The results suggest that delayed harvesting could increase FB1 contamination in maize. SHORT COMMUNICATION In 2005-2007, vineyards affected by esca disease were surveyed in the provinces of Alicante and Valencia (south-east of Spain). The presence of resupinate, hymenochaetaceous basidiocarps was observed on the trunks of vines that displayed typical esca symptoms as well as in apparently healthy plants. Vines showing fruiting bodies represented 40% of the total esca-diseased plants and were randomly distributed in the several vineyards surveyed. Using classical and molecular identification methods, the fungus producing the observed basidiocarps was recognized as Inonotus hispidus, whose pathogenicity was tested by in vitro inoculation of healthy vines with fresh inoculum of six different isolates. Although I. hispidus has been previously reported from Vitis spp., to our knowledge this is the first record of the presence of its fruiting bodies on esca-diseased vines in Spain. SHORT COMMUNICATION Seventyfive monoconidial isolates of Magnaporthe grisea species complex were obtained from rice and some grasses from north Iran during 1997-2005. Thirtyfive isolates were from rice and 40 from crabgrass, foxtail millet, barnyard millet, and some unknown weeds. All these isolates and eight standard mating type tester isolates were analyzed with complementation tests using nitrate non-utilizing (nit) mutants to determine vegetative compatibility groups (VCGs) and genetic relationship between the two groups of isolates. All rice isolates were grouped into four VCGs designated as VCG1, VCG2, VCG3, and VCG4. VCG3 was the most common group and included 14 isolates, whereas the isolates obtained from grasses belonged to three different multimember VCGs, denoted VCG5, VCG6, and VCG7. VCG5 included 29 isolates out of 40 and other two multimember VCGs contained four isolates each. Results showed that although Magnaporthe populations infecting rice and gramineae weeds share a common ancestry, no vegetative compatibility was observed between them. This study also indicated that there is no correlation between field isolates and standard mating type isolates. Thus, determination of VCGs is a useful mean for characterizing genetic diversity within and among all Magnaporthe species from different hosts. SHORT COMMUNICATION We tested the transmission efficiency of two genetically divergent isolates of Broad bean wilt virus 1 (BBWV- 1), PV-132 from the USA, and Ben from Spain, by two aphid species, Myzus persicae (Sulzer) and Aphis gossypii (Glover) collected in Spain. Efficiency was estimated as the number of infected plants divided by the number of single-aphid-inoculated plants. The two aphid species transmitted the virus isolates with equivalent efficiency, but the transmission rate was significantly higher for isolate Ben than for PV-132. It is hypothesized that the specificity determinants for vector-virus interaction might be similar for M. persicae and A. gossypii, whereas transmission rate could be affected by amino acid differences in the coat proteins of the two isolates. SHORT COMMUNICATION A grapevine accession of cv. Mavro from Cyprus showing mild leafroll symptoms and containing closterovirus- like virus particles failed to react with commercial antisera to Grapevine leafroll-associated virus 1, - 2, -3, -5 and -7. The virus, denoted GLRaV-Cyp1, was not mechanically transmissible to herbaceous hosts but was successfully transferred by chip-bud grafting and through Planococcus ficus to healthy rooted cutting of cv. Cabernet sauvignon in which it induced leafroll symptoms as mild as those seen in the mother vine. Three different fragments of the viral genome, corresponding to the heat shock protein 70 homologue (HSP70h), major coat protein (CP) and p23 genes were amplified using degenerate and specific primers, cloned and sequenced. In a comparative analysis, GLRaV-Cyp1 showed amino acid sequence identity never higher than 86% with the HSP70h and 70% with CP and p23 genes of other GLRaVs species and strains, with the exception of a GLRaV isolate from a Greek grapevine, with which it showed 94% identity. In phylogenetic trees constructed with sequences of HSP70h, CP and p23 genes, GLRaV-Cyp1 consistently grouped in a cluster comprising sequences of the “type strains” of GLRaV-4, -5, -6, and -9 and of several other viral isolates regarded as molecular variants of one or more of the above species. This supports the notion that GLRaV-Cyp1 may not be a new ampelovirus species. In a small-scale survey carried out by PCR on a collection of vines from ten Mediterranean countries, GLRaVCyp1 was detected in 5 out of 121 vines tested. SHORT COMMUNICATION Citrus leaf blotch virus (CLBV), the type species of the putative new genus Citrivirus, causes a bud union disorder of Nagami kumquat and Calamondin scions grafted on trifoliate rootstocks. This virus was successfully transmitted to Nicotiana cavicola using leaf extracts of infected Nagami kumquat and Etrog citron, thus widening its herbaceous host range. The infection was latent but confirmed by the positive response of RTPCR using virus-specific primers and by electron microscopy. The positive transmission of CLBV to N. cavicola should in principle facilitate laboratory investigations, as it provides a new source of virus alternative to and more manageable than citrus. SHORT COMMUNICATION Grapevine fanleaf virus (GFLV), Grapevine leafroll associated virus 1, 2, and 3 (GLRaV-1,-2, and -3), Grapevine virus A (GVA) and Grapevine fleck virus (GFkV) were monitored monthly throughout a year in naturally infected field-grown vines by ELISA and RTPCR. The organs tested were: opening buds in September, tips or unfolded leaves in September and October, leaf petioles from October to April, completely expanded leaves from November to April, green phloem tissues from October to February and cortical scrapings from December to August. Phloem of lignified canes, when available, is the best source for all viruses tested, allowing 100% detection by ELISA and RT-PCR. This is the first study carried out in South America to establish the best plant material and sampling times to optimize grapevine virus detection by ELISA and RT-PCR. SHORT COMMUNICATION A multiplex PCR assay for the simultaneous detection of three bacterial seed-borne pathogens of tomato was developed. Published primers: (i) CMM-5-CMM-6 for Clavibacter michiganensis pv. michiganensis; (ii) primer 1- primer 2 for Pseudomonas syringae pv. tomato; (iii) RST2- RST3 for Xanthomonas axonopodis pv. vesicatoria, were used in the assay. Annealing temperatures were determined by gradient PCR individually for each pathogen and primer concentration ratios were investigated. Sensitivity assays were carried out and compared with single PCR. Temperature of 59±1°C was optimal for annealing. Optimal primer concentrations were determined as 0.36 μMol l-1 for C. m. michiganensis, 0.30 μMol l-1 for X. a. vesicatoria, and 0.12 μMol l-1 for P. s. tomato. Sensitivity assays showed that 3 CFU in 50 μl sterile distilled water, derived from pure cultures of C. m. michiganensis, P. s. tomato and X. a. vesicatoria could reliably be detected by multiplex PCR when applied to pure cultures. The detection limit was determined to be ca. 10 times lower than that of single PCR. Multiplex PCR provided less labor and rapid results for the detection of bacterial pathogens of tomato, but the sensitivity of detection was reduced. Thus, the sensitivity of this technique should be assayed prior to its use in place of single PCR.
|
|
|
||||||||||||||||||||||||||||
 |
||||||||||||||||||||||||||||||||
|
|
|
|
|
||||||||||||||||||||||||||||
