Showing papers in "Journal of Phytopathology in 2007"

Biological Control of Phytophthora Root Rot of Pepper Using Trichoderma harzianum and Streptomyces rochei in Combination

Abstract: A combination of two compatible micro-organisms, Trichoderma harzianum and Streptomyces rochei, both antagonistic to the pathogen Phytophthora capsici, was used to control root rot in pepper. The population of the pathogen in soil was reduced by 75% as a result. Vegetative growth of the mycelium of P. capsici was inhibited in vitro on the second day after P. capsici and T. harzianum were placed on the opposite sides of the same Petri plate. Trichoderma harzianum was capable of not only arresting the spread of the pathogen from a distance, but also after invading the whole surface of the pathogen colony, sporulating over it. Scanning electron microscopy showed the hyphae of P. capsici surrounded by those of T. harzianum, their subsequent disintegration, and the eventual suppression of the pathogen's growth. Streptomyces rochei produced a zone of inhibition, from which was obtained a compound with antioomycete property secreted by the bacteria. When purified by high-pressure liquid chromatography, this compound was identified as 1-propanone, 1-(4-chlorophenyl), which seems to be one of the principal compounds involved in the antagonism. A formulation was prepared that maintained the compound's capacity to inhibit growth of the pathogen for up to 2 years when stored at room temperature in the laboratory on a mixture of plantation soil and vermiculite. The two antagonists, added as a compound formulation, were effective at pH from 3.5 to 5.6 at 23-30°C. The optimal dose of the antagonists in the compound formulation was 3.5 x 10 8 spores/ml of T. harzianum and 1.0 x 10 9 FCU/ml of S. rochei. This is the first report of a compound biocontrol formulation of these two antagonists with a potential to control root rot caused by P. capsici.

Influence of growth stage on fusarium head blight and deoxynivalenol production in wheat

Abstract: Fusarium head blight is a major concern for wheat production worldwide. The fungi that cause the disease may infect head tissues from flowering to late stages of kernel development, but a better understanding of the influence of the time of infection on grain weight reduction and mycotoxin accumulation resulting from the infection process is needed. We investigated the influence of wheat reproductive stage at the time of inoculation on disease and grain quality parameters, especially production of deoxynivalenol (DON) in mature grains. Heads of Norm wheat were spray inoculated with a macroconidial suspension of a DON-producing isolate of Fusarium graminearum at each of six reproductive stages from flowering to hard dough. Plants were incubated in a mist chamber for 48 h and then moved to the greenhouse until maturity. Norm wheat was susceptible at all stages inoculated but the highest grain weight reduction and DON accumulation occurred in plants inoculated past flowering to late milk stages. However, high incidences of kernel infection and significant levels of DON accumulation resulted from inoculations as late as the hard dough stage, even though there was no corresponding reduction in grain weight compared to non-inoculated plants. The occurrence of commercially significant levels of DON in plump, high-yielding wheat may result from infections that occur during favourable environments well after the flowering stages. Late infection and DON production should therefore be a future research focus for wheat breeding and integrated management of FHB and an important consideration for grading systems that employ the presence of visibly damaged kernels as a means of estimating DON content of wheat.

Plant Resistance to Pathogen Infection: Forms and Mechanisms of Innate and Acquired Resistance

Abstract: Different forms and mechanisms of plant resistance are compared in this review, differentiating between innate and acquired resistance. Within innate resistance, general non-specific and specific host resistance are treated as regards the phenomena and mechanisms. Various forms of non-specific (e.g. non-host, basal, quantitative etc.) as well as specific resistance (extreme resistance, gene-for-gene resistance with the hypersensitive response, toxin resistance and gene silencing) are discussed. Within acquired resistance, the immune memory vs. stress memory of animal or plant hosts are compared. Furthermore, mechanisms of acquired resistance of plants are treated in relation to salicylate metabolism, systemic signalling, antioxidants and a recently discovered recombination signal inducing somatic mutations. It seems important for future research trends in plant pathology that several new resistance mechanisms have been discovered or at least re-evaluated, in addition to the specific HR-associated resistance, which has been the subject of the majority of previous research.

Pathotype Classification of Plasmodiophora brassicae and its Occurrence in Brassica napus in Alberta, Canada

Abstract: A field survey for clubroot of crucifers, caused by Plasmodiophora brassicae, was conducted in the regions surrounding Edmonton, Alberta, Canada, in 2005. The presence of clubroot was confirmed in 41 of the 112 canola (Brassica napus) fields surveyed. These P. brassicae- infested fields were located in Sturgeon, Strathcona, Leduc and Wetaskiwin counties, as well as in a rural area of northeast Edmonton. Infected roots were also received from an infested field in Flagstaff County, southeast of Edmonton. Although there was a significant negative correlation between index of disease and soil pH, the occurrence of clubroot was not restricted to fields with acidic soils. Populations of the pathogen were selected from 10 fields and used in pathotype classification on the differential hosts of Williams, Some et al. and the European Clubroot Differential (ECD) set. Kruskal-Wallis analysis indicated no significant differences in the virulence of the 10 populations tested, suggesting that they are relatively homogenous. If a disease index of 50% was regarded as the cut-off between a resistant and a susceptible reaction, then all P. brassicae populations tested were classified as ECD -/15/12 on the hosts of the ECD set, or as pathotypes 3 or P2 on the differentials of Williams or Some et al. respectively. However, it may be difficult to detect rare or infrequent pathotypes when field populations of the pathogen are used for characterization.

Microbial fungicides in the control of plant diseases

Abstract: As environmental and commercial requirements for new fungicides are increasingly demanding, antifungal compounds of microbial origin attract tremendous interest as a starting point in the development of environmentally sound agricultural fungicides. As seen in fenpiclonil, fludioxonil and synthetic derivatives of the strobilurins such as azoxystrobin and krexosim-methyl, this approach for the development of microbial fungicides has proven to be a promising and effective strategy for developing new fungicides. As a result, microbial metabolites face a revival as lead compounds. Recently, numerous antifungal compounds were discovered from diverse microbial sources using traditional activity-based screening techniques. These microbial compounds showed potent control efficacy against various plant diseases, including chronic diseases which are difficult to control with conventional synthetic fungicides. Advances in screening systems directed to specific targets of fungal metabolism have increased the opportunities to discover novel antifungal agents with selectivity over non-target organisms. Microbial metabolites have also been exploited as a source for non-fungicidal disease control agents that do not inhibit vegetative hyphal growth, but rather interfere specifically with the infection process of pathogenic fungi. The specificity of microbial fungicides is a highly preferred characteristic in terms of impacting the environment, where it is closely related to the occurrence of fungicide resistance. The most recently developed fungicides from microbial metabolites, the strobilurins, provide a cue for the high risk of resistance development of site-specific fungicides.

New Pythium Taxa Causing Root Rot on Mediterranean Quercus Species in South-west Spain and Portugal

Abstract: Pythium spiculum, a recently described new taxon, has been frequently isolated from declining Quercus rotundifolia and Q. suber roots and rhizosphere since 2003 in southern Iberia. In soils of declining Quercus forests this species was found as frequently as Phytophthora cinnamomi which, until now, was the only oomycete described as a Quercus root rot pathogen in the region. Inoculation tests conducted on young Q. rotundifolia plants showed that Py. spiculum is an aggressive root pathogen, although producing severities of symptoms significantly lower than those of P. cinnamomi. This new pathogen could play a role as decline factor in southern Iberia. Another new species, Py. sterilum, was also found to be pathogenic to Quercus roots but there are presently only few records of this organism isolated from rhizosphere of declining oaks in central Spain. More than an active decline factor, this species should be considered as a potential risk for Quercus forests.

Effect of Seed Treatment with Rhizobium leguminosarum on Pythium Damping-off, Seedling Height, Root Nodulation, Root Biomass, Shoot Biomass, and Seed Yield of Pea and Lentil

Abstract: Field experiments were conducted in 2004 and 2005 to determine the effects of seed treatment with Rhizobium leguminosarum bv. viceae on damping-off, seedling height, root nodule mass. root biomass, shoot biomass and seed yield of pea and lentil in a field naturally infested with Pythium spp. Compared with the untreated controls, treatment of pea seeds with R. leguminosarum bv. viceae strains R12, R20 or R21 significantly (P < 0.05) reduced incidence of damping-off, promoted seedling growth and increased root nodule mass, root biomass and shoot biomass. Seed treatments with R12 or R21 also resulted in a significant (P < 0.05) increase in seed yield of pea. The strain R21 was most effective among the four strains of R. leguminosarum bv. viceae tested in peas. Although, the level of disease control by strain R21 was similar to seed treatment with the fungicide Thiram TM , R21 was more effective in enhancing root nodule production and promoting plant growth. For lentil, treatment of seeds with R. leguminosarum bv. viceae strains R12 or R21 significantly (P < 0.05) reduced incidence of damping-off compared with the untreated control. All of the four strains of R. leguminosarum bv. viceae tested increased lentil seedling height. root nodule mass and shoot biomass, and all except R20 increased root biomass. Seed yield was higher for the treatments of R12 and R21. The strain R12 was most effective among the four strains of R. leguminosarum bv. viceae tested in lentil. Although, strain R12 was as effective as Thiram TM for control of damping-off of lentil, it was more effective than Thiram TM for the production of root nodules and promotion of plant growth. The study concludes that seed treatment with R. leguminosarum bv. viceae is effective in control of Pythium damping-off of pea and lentil and that the efficacy of control is strain specific, strain R21 for control of the disease on pea and strain R12 for control of the disease on lentil.

Detection of Strawberry RNA and DNA Viruses by RT-PCR Using Total Nucleic Acid as a Template

Abstract: Strawberry mild yellow edge virus (SMYEV), Strawberry mottle virus (SMoV) and Strawberry vein banding virus (SVBV), three of strawberry viruses can cause a severe loss in commercial strawberry worldwide. In this paper, total nucleic acid from strawberry leaves was compared with total RNA as the template for detection of the above viruses both by single and multiplex reverse transcription polymerase chain reaction (RT-PCR). Total nucleic acid extract with the modified CTAB method and total RNA extracts with both the modified RNeasy method and the modified CTAB method examined by electrophoresis resulted in a high quality ribosomal 28S and 18S RNA when loaded on an agarose gel. When total RNA and total nucleic acid were used as templates, detection results of SMYEV and SMoV were identical and the amplified band intensity was nearly the same whether by single or duplex RT-PCR. Using total nucleic acid as the template, the detection results of SMYEV and SMoV by multiplex RT-PCR were in a good concord with that amplified by single and duplex RT-PCR, and for SVBV this was similar to the result of detection that amplified by direct PCR using pure DNA as the template. This study demonstrates that total nucleic acid extracted from strawberry leaves by a modified CTAB method is suitable as a template for virus detection by RT-PCR. This makes investigation of the epidemiology of strawberry viruses much easier.

Analysis of the Antimicrobial Activity of Flavonoids and Saponins Isolated from the Shoots of Oats (Avena sativa L.)

Abstract: Crude extracts from the shoots of oat cv. Quoll were tested against four species of bacteria and eight species of fungi. Bacterial growth was not inhibited. The mycelia growth of all Pyrenophora species tested, except Pyrenophora avenae DAR 33699, was inhibited by the crude extract, whereas the mycelia growth of Fusarium graminearum, Mycosphaerella pinodes and Rhizoctonia solani was not affected. Partially purified fractions with high concentrations of flavone-C-glycosides had no inhibitory effect against P. teres f. sp. teres and P. teres f. sp. maculata. The saponin 26-desglucoavenacoside B accounted for most of the activity against P. teres f. sp. teres with a minor contribution from the other saponins, 26-desglucoavenacoside A and avenacosides A and B.

Morphological and Pathological Variability in Rice Isolates of Rhizoctonia solani and Molecular Analysis of their Genetic Variability

Abstract: Nineteen isolates of Rhizoctonia solani collected from different rice varieties grown in various regions of Pun-jab were studied for their morphological and pathological characterization. Majority of the isolates were fast growing with raised and fluffy colonies and hyphal width of 9.6 μm while four exhibited moderate growth rate. Colony colour in all except two isolates was light yellowish brown. While sclerotial number per 5.0 mm culture disc of the test isolates ranged between 2.1 and 11.2 mm, their size varied between 1.31 and 2.08 mm. Sclerotial colour in all except two isolates was dark brown and most of these were found scattered in the colony. There was no relationship between morphologically similar isolates and their pathogenic behaviour. Majority of the isolates produced lesion length between 45.6 and 58.2 mm on detached rice leaves (cv. PR116). Molecular characterization of genetic diversity in the test isolates was studied by using 10 inter simple sequence repeats (ISSR) and eight random amplified polymorphic DNA (RAPD) markers. The size of amplified DNA bands ranged from 0.25-3.0 to 0.5-4.0 kb with ISSR and RAPD markers, respectively. Combined data set of 155 DNA markers were analysed with UPGMA resulting five clusters with 49-89% genetic similarity. Most of the isolates showed grouping specific to the host variety. Out of these two types of DNA markers, RAPD markers were able to detect more genetic variability when compared to ISSR markers.

Characterization of Colletotrichum lindemuthianum Isolates from the State of Minas Gerais, Brazil

Abstract: Anthracnose disease of common bean (Phaseolus vulgaris), caused by Colletotrichum lindemuthianum, is responsible for extensive yield losses worldwide. This pathogen is known to vary greatly in its pathogenicity. Control strategies include chemical control and, mainly, the development of resistant cultivars, taking into account the population structure of C. lindemuthianum. The objective of this study was to investigate the pathogenic and genetic diversity and population structure among C. lindemuthianum isolates collected in Minas Gerais state, Brazil. When these isolates were inoculated on 12 differential cultivars, a total of 10 races were identified within a series of 48 isolates collected in Minas Gerais, Brazil. Races 65, 81 and 73 were the most frequent races and occurred in most of the regions. This study also detected race 337, which had not been reported previously in the literature. Random amplified polymorphic DNA (RAPD) analysis performed on the same 48 isolates revealed great genetic diversity, clustering the series into five groups at a maximum similarity value of 89.6%. There was no clear relationship between the loci sampled by RAPD markers and the pathogenic characterization. Analysis of molecular variance showed that 96.06% of the variability was contained within regions and 3.94% among regions, indicating a high exchange of genetic material among the regions of the State. Most of the variability was detected within races (75.24%). The pathogenicity and RAPD assays corroborated the broad genetic diversity of the pathogen and the results have been useful in breeding for resistance to anthracnose.

Ability of moderately halophilic bacteria to control grey mould disease on tomato fruits

Abstract: Tomato is one of the leading crops in Tunisia in terms of weight consumed (20 kg/per person/year). Preserving the quality of the fruit from field to consumer is essential to successful marketing. Grey mould rot induced by Botrytis cinerea is an important cause of postharvest loss depending on season and handling practices. We describe here the ability of halotolerant to moderately halophilic bacteria isolated from different Tunisian Sebkhas (hypersaline soils) to protect fresh-market tomato fruits from B. cinerea. The tomatoes tested were at two different stages of ripening, (i) mature-green and (ii) red. Six strains significantly reduced growth of the pathogens from 67% to 87%. The effectiveness of these antagonists was also confirmed on green tomatoes; in which the fruit rot protection rate ranged from 74% to 100%. The antagonists were characterized by morphological, biochemical and physiological tests as well as 16S rDNA sequencing. The halotolerant effective isolates were identified as belonging to one of the species Bacillus subtilis (M1-20, J9) or B. licheniformis (J24). One effective moderately halophilic isolate (M2-26) was identified as Planococcus rifietoensis. These strains are a source of hydrolytic enzymes such as chitinases, proteases, laminarinases, amylases, lipases and cellulases. For comparison, 12 halotolerant or moderately halophilic strains obtained from DSM culture collection were also evaluated for their antifungal activity against B. cinerea on tomato fruits. The most effective strains were Halomonas subglaciescola, Halobacillus litoralis, Marinococcus halophilus, Salinococcus roseus, Halovibrio variabilis and Halobacillus halophilus with a percentage of grey mould rot reduction ranging from 71% to 97%. Inoculation of mature-green tomatoes by the bacterial antagonist of Halobacillus trueperi resulted in no disease development. Our results indicate that the use of halotolerant to halophilic micro-organisms should be helpful in reducing grey mould disease of stored tomatoes.

The Occurrence and Characterization of New Recombinant Isolates of PVY Displaying Shared Properties of PVYNW and PVYNTN

Abstract: Isolates of Potato virus Y (PVY), collected in Syria, induced vein necrosis in tobacco but reacted to PVY° monoclonal antibody, and were therefore classified as PVY N W (or PVY N:O ). The possible recombinant points within the genome were checked using a previously reported multiplex RT-PCR. These isolates were categorized in two groups according to recombinant points. Isolates with one recombinant point at the HC-Pro/P3, identical to previously reported PVY N W (or PVY N:O ), were designated as group 1. However, the majority of Syrian PVY isolates showed two recombinant points at both HC-Pro/P3 and 6K2/NIa, revealing a new genomic recombination pattern that had never been reported for PVY N W (or PVY N:O ), hereafter referred to as group 2. Further characterizations showed that isolates of group 2 had an additional recombinant point at the N-terminal of the coat protein gene (CP) of their genome. Based on the bioassay on tobacco, serological and molecular studies, these isolates showed shared characteristics of both PVY N W and the recombinant PVY NTN , making it difficult to include them in any of these variants. We propose them as new isolates of PVY N strain, and label them PVY SYR (SYR is from Syria, the country of origin).

Determination of Deoxynivalenol and Nivalenol Chemotypes of Fusarium graminearum Isolates from China by PCR Assay

Abstract: Fusarium graminearum is the main causative agent of cereal scab and maize cob rot in China. Two hundred and fifty-five F. graminearum isolates were obtained from wheat, barley and maize from Hebei, Heilongjiang and Hubei, provinces with distinct climate conditions and cropping systems. The isolates were confirmed to be F. graminearum by polymerase chain reaction (PCR) assay using F. graminearum species-specific primers Fg16F/Fg16R. Two populations, 7C1 and 6A5, were identified to exist in China. The 6A5 population was predominant in Hubei in central China along the Yangtze River, whereas the 7C1 population was predominant in Heilongjiang and Hebei in northern China. Based on sequences of Tri13 and Tri3, genes involved in the mycotoxin biosynthetic pathway, PCR assays were used to detect 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON) and nivalenol (NIV) chemotypes. All three chemotypes of F. graminearum were identified, with 15-ADON chemotype predominating overall. A greater proportion of 3-ADON chemotype was found in Hubei, whereas a greater proportion of 15-ADON was found in Heilongjiang and Hebei.

Colonization of Female Watermelon Blossoms by Acidovorax avenae ssp. citrulli and the Relationship between Blossom Inoculum Dosage and Seed Infestation

Abstract: The aim of this work was to investigate the ability of Acidovorax avenae ssp. citrulli, the causal agent of bacterial fruit blotch of cucurbits (BFB), to colonize female watermelon blossoms, and to explore the relationship between blossom inoculum dosage and seed infestation. Under greenhouse conditions A. avenae ssp. citrulli colonized stigmas and styles of female watermelon blossoms reaching populations of [almost equal to]10⁷ to 10⁸ colony-forming units (CFU) per blossom for 96 h after inoculation. Acidovorax avenae ssp. citrulli growth on stigmas was slower than that of Pseudomonas syringae Cit7, a non-pathogenic, foliar epiphyte of tomato. While pollination reduced growth of A. avenae ssp. citrulli, but P. syringae Cit7 was unaffected. Both bacteria colonized style tissues but bacterial growth in the style was significantly less than the stigma. Blossom inoculation with [almost equal to]1 x 10³A. avenae ssp. citrulli CFU/blossom led to 36-55% infested seedlots within symptomless fruits. On average 14% of the seedlings produced from these seedlots displayed BFB symptoms. There was a strong positive correlation between A. avenae ssp. citrulli inoculum concentration applied to blossoms and the percentage of infested seedlots, as determined by the seedling grow-out assay (R² = 0.94). However, this relationship was weaker when seedlot infestation was determined by a polymerase chain reaction-based assay (R² = 0.34). There was also a strong positive linear relationship between A. avenae ssp. citrulli blossom inoculum dose and the mean percentage of BFB-infected seedlings (R² = 0.99) produced in seedling grow-out assays. These data support the hypothesis that blossom colonization might be involved in seed infestation under field conditions.

Detection of Aflatoxin in Aspergillus Species Isolated from Pistachio in Iran

Abstract: To estimate the incidence contamination of fresh pistachio nuts by aflatoxigenic fungi in Iran, nut samples were collected from pistachio orchards in Kerman, Rafsanjan and Isfahan regions. Out of the 200 Aspergillus isolates obtained, 11 species were identified as A. alliaceous, A. candidus, A. flavus, A. niger, A. niveus, A. ochraceus, A. parasiticus, A. tamari, A. terreus, A. unguis and A. wentii. For detection of aflatoxin production ability of the isolates, three target genes, namely aflR, aflJ, and omtB, used in PCR amplification. In all the examined cases, the degenerate primer designed for amplification of omtB gene, named omtBII, was able to amplify an expected 611 bp fragment in aflatoxigenic isolates in this study and yielded the same result as those obtained from TLC analysis and fluorescence ability by application of methylated b-cyclodextrin in culture media. Using this procedure the significant incidence of aflatoxin-producing aspergilli was confirmed in pistachio nuts produced in different regions of Iran. The results indicated that PCR method described here, in combination with fluorescence assay, is a reliable and simple confirmatory test for monitoring pistachio nuts contaminated with aflatoxinogenic aspergilli.

Development and Validation of Conventional and Quantitative Polymerase Chain Reaction Assays for the Detection of Storage Rot Potato Pathogens, Phytophthora erythroseptica, Pythium ultimum and Phoma foveata

Abstract: The diseases pink rot, watery wound rot and gangrene are important storage rot diseases of potato associated predominantly with Phytophthora erythroseptica (P.), Pythium ultimum (Py.) and Phoma exigua (Phoma) var. foveata respectively. Reliable molecular-based diagnostic tests are required that will not only allow unequivocal identification of symptoms but will further advance epidemiological studies of these potato diseases to increase our understanding and contribute to more effective management and control strategies to the potato industry. Primers and probes were designed in specific regions of the internal transcribed spacer (ITS) regions to develop conventional and real-time quantitative polymerase chain reaction (PCR) assays able to detect all possible fungal and oomycete pathogens causing pink rot, watery wound rot and gangrene. The specificity of each diagnostic assay was rigorously tested with over 500 fungal/ oomycete plant pathogen isolates from potato and reference culture collections, and both conventional and real-time PCR methods produced similar results. In terms of sensitivity, the detection limits for real-time PCR went below ag DNA levels compared with pg DNA levels with conventional PCR. The real-time PCR assays developed to detect Phoma foveata and Py. ultimum on tubers were suitable for the comparative C t method (∇∇Ct) of quantification using the cytochrome oxidase gene of potato as a normalizer assay; an advantage as the need for a standard curve is eliminated. Each assay detected Phoma species (var. foveata or exigua) from naturally infected tubers showing symptoms of gangrene, and P. erythroseptica or Py. ultimum were also detected following inoculation of Russet Burbank tubers. Each diagnostic assay developed could reliably detect and distinguish between the pink rot, watery wound rot and gangrene-causing potato pathogens.

Natural Infection by Tospoviruses of Cucurbitaceous and Fabaceous Vegetable Crops in India

Abstract: Natural infection of tospoviruses on three cucurbitaceous (Cucumis sativus, cucumber; Luffa acutangula, ridge gourd; Citrullus lanatus, watermelon) and three fabaceous (Vigna unguiculata, cowpea; Phaseolus vulgaris, French bean; Dolichos lablab, sem) vegetable crops in India was identified on the basis of nucleocapsid protein (NP) gene characteristics. The complete NP gene of the cowpea isolate from Kerala and the sem isolate from Tamil Nadu was 831 nucleotides long, encoding a protein of 276 amino acids. For other Tospovirus isolates from cucumber, French bean, ridge gourd and watermelon, the partial NP gene (291 nt) was sequenced. Comparative NP gene sequence analyses revealed that fabaceous isolates shared maximum identity both at the nucleotide (92–97%) and amino acid (93–97%) levels with the corresponding region of Groundnut bud necrosis virus (GBNV), whereas cucurbitaceous isolates shared maximum identity both at nucleotide (93–99%) and amino acid (95–98%) levels with the corresponding region of Watermelon bud necrosis virus (WBNV), results suggesting that the Tospovirus isolates infecting fabaceous hosts should be regarded as strain of GBNV, whereas those infecting cucurbitaceous hosts as a strain of WBNV. Nucleocapsid protein gene was conserved both in GBNV and WBNV isolates originating from different hosts and locations.

Microsatellite and Morphological Markers Reveal Genetic Variation within a Population of Sclerotinia sclerotiorum from Oilseed Rape in the Çanakkale Province of Turkey

Abstract: The genetic variation among a population of Sclerotinia sclerotiorum collected from oilseed rape fields in the Canakkale Province of Turkey was assessed using molecular and morphological markers. Seven microsatellite primer pairs (out of eight) revealed 32 clear polymorphic alleles among the 36 fungal isolates examined. An unweighted pair-group mean analysis dendrogram was generated using the genetic distance matrix with the 32 microsatellite alleles. The level of similarity was as low as 15% between some isolates indicating a high level of genetic diversity within the fungal population; 23 distinct isolates were found (at a genotypic diversity level of 63%). Among the collection of 36 isolates, 19 mycelial compatibility groups (MCGs) were identified; 10 MCGs included at least two isolates. Molecular and morphological data suggest that most of the isolates within a single MCG were identical; however, the isolates belonging to the MCG2 and MCG4 had variable microsatellite haplotypes and were morphologically dissimilar. The data suggest that there is possibly a high rate of outcrossing as well as evolutionary potential within the population of the pathogen in oilseed rape fields. This is the first report demonstrating the genetic and morphological variation within a population of S. sclerotiorum in Turkey.

Pathogenic Fungi in Garlic Seed Cloves from the United States and China, and Efficacy of Fungicides Against Pathogens in Garlic Germplasm in Washington State

Abstract: Commercially distributed garlic (Allium sativum) seed cloves from six states of the United States and mainland China were surveyed for the presence of fungi recorded as pathogenic to garlic in the literature. Aspergillus niger, A. ochraceus, Botrytis porri, Embellisia allii, Fusarium oxysporum f. sp. cepae, F. proliferatum and Penicillium hirsutum, were each recovered from one or more of these commercial sources, as was F. verticillioides, not previously reported as pathogenic to garlic, but here demonstrated to be a pathogen. Seed garlic distributed from public germplasm collections may also contain fungal pathogens: E. allii, F. oxysporum f. sp. cepae and/or F. proliferatum caused severe losses in 2002-2003 and 2005-2006 during germplasm regeneration and storage for the National Plant Germplasm System in Pullman, Washington. Use of fludioxonil, thiophanate methyl and/or benomyl (the latter withdrawn from the market, but used here as a standard) at label rates against E. allii and Fusarium species promoted plant health, but not when infections were located deep within tissues nor under some situations involving high disease pressure.

Microsatellite Markers Associated with Spot Blotch Resistance in Spring Wheat

Abstract: Spot blotch, caused by Cochliobolus sativus, is a serious wheat (Triticum aestivum L.) disease in the warm areas of South Asia. Breeding for resistance in the past 15 years has produced limited progress, and newly developed wheat cultivars suffer considerable yield reductions under spot blotch epidemics in the region. Resistance is often controlled by multiple genes with additive effects. Marker-assisted selection, in combination with field selection, could accelerate the identification of progeny with multiple genes for resistance early in the breeding process. A study was conducted to determine microsatellite markers associated with resistance in the F 7 progeny from a cross between the spot blotch-susceptible Sonalika and resistant G162 wheat genotypes. A parental survey using 171 simple sequence repeats (SSR) primer sets and spread over 21 chromosomes of wheat identified 52% polymorphic loci. However, only 15 polymorphic markers showed association with two bulks, one each of progeny with low and with high spot blotch severity. The detailed analysis indicated that progeny lines with low spot blotch severity could be separated from those with high severity using three SSR markers located on three wheat chromosomes. The findings may be useful in developing a marker-assisted selection strategy for spot blotch resistance in wheat.

Biocontrol and Plant Pathogenic Fusarium oxysporum-Induced Changes in Phenolic Compounds in Tomato Leaves and Roots

Abstract: The biocontrol fungus Fusarium oxysporum strain CS-20 was previously shown to reduce the incidence of Fusarium wilt of tomato through an uncharacterized host-mediated response. As phenolic compounds are involved in the defence response of tomato to pathogens and other stressors, this work was undertaken to determine whether biocontrol strains induced changes in phenolic compounds in leaves and roots of tomato seedlings in the presence and absence of pathogenic F. oxysporum f. sp. lycopersici. Roots of intact tomato seedlings were placed in water or aqueous fungal spore suspensions. Two biocontrol F. oxysporum strains [CS-20 (host-mediated mechanism) and 85SK-1 (control mechanism unknown)] and two plant pathogenic strains of F. oxysporum f. sp. lycopersici Race 1 were used. After 24 or 72 h exposure, phenolic compounds were extracted from leaves and roots before identification by HPLC. There were significant qualitative and quantitative differences between the two sampling times. Compared with the control treatment, strain CS-20 significantly altered (usually increasing) the ferulic, caffeic and vanillic acid contents, and concentrations once unidentified phenolic compounds recovered from leaves and roots. In another experiment, tomato seedlings growing in sterile sand were drenched with spores of strain CS-20 the day before treating them with varying concentrations of spores of the pathogen for 24 or 72 h. The amount of pathogen present did not significantly affect the plant phenolic response to the presence of strain CS-20. This work demonstrates that tomato responds within 24 h to the presence of the biocontrol strain CS-20 by alterations in secondary metabolism that are typical of resistance responses in tomato.

Detection and Characterization of Phytoplasmas Infecting Ornamental and Weed Plants in Iran

Abstract: During field surveys in 2004, ornamental and weed plants showing symptoms resembling those caused by phytoplasmas were observed in Mahallat (central Iran). These plants were examined for phytoplasma infections by polymerase chain reaction (PCR) assays using universal phytoplasma primers directed to ribosomal DNA (rDNA). All affected plants gave positive results. The detected phytoplasmas were characterized and differentiated through restriction fragment length polymorphism (RFLP) and sequence analysis of PCR-amplified rDNA. The phytoplasmas detected in diseased Asclepias curassavica and Celosia argentea were identified as members of clover proliferation phytoplasma group (16SrVI group) whereas those from the remaining plants examined proved to be members of aster yellow phytoplasma group (16SrI group) (‘Candidatus Phytoplasma asteris’). In particular, following digestion with AluI, HaeIII and HhaI endonucleases, the phytoplasma detected in Limonium sinuatum showed restriction profiles identical to subgroup 16SrI-C; phytoplasmas from Gomphocarpus physocarpus, Tanatacetum partenium, Lactuca serriola, Tagetes patula and Coreopsis lanceolata had the same restriction profiles as subgroup 16SrI-B whereas Catharanthus roseus- and Rudbeckia hirta-infecting phytoplasmas showed restriction patterns of subgroup 16SrI-A. This is the first report on the occurrence of phytoplasma diseases of ornamental plants in Iran.

A TaqMan PCR Method for Routine Diagnosis of the Quarantine Fungus Guignardia citricarpa on Citrus Fruit

Abstract: With respect to disease risk for the quarantine fungus Guignardia citricarpa on citrus fruit an accurate diagnosis for routine analysis is required. Also, when inspections have to be performed on imported citrus fruits, a fast detection method is urgently needed. A fast automated DNA extraction method based on magnetic beads combined with a real-time PCR assay was optimized to improve and advance the routine diagnosis of citrus black spot disease. Real-time PCR was used for detection of the pathogen G. citricarpa in planta. A specific primer/TaqMan probe combination that discriminates between G. citricarpa and the harmless citrus endophyte Guignardia mangiferae, was designed based on the internal transcribed spacer region of the multi-copy rDNA gene. Co-amplification of target DNA along with an internal competitor DNA fragment made the diagnostic assay more reliable to check for false negatives. The real-time PCR was specific, since no cross reaction was observed with a series of citrus pathogens and related species. The diagnostic assay was performed on lesions dissected from imported diseased oranges. Comparison between the conventional PCR and the real-time PCR methods showed that the TaqMan method was more sensitive.

Virulence Analysis and Race Classification of Xanthomonas oryzae pv. oryzae in China

Abstract: Two hundred and eighty-five isolates of Xanthomonas oryzae pv. oryzae were randomly collected from 22 rice-growing provinces in China. Ninety-one representative isolates were chosen to assess the differential characteristics of 24 near-isogenic rice lines containing a single resistance gene or two to four genes. Most isolates were avirulent on pyramided lines, except IRBB51, and hence, the pyramided lines cannot be used as differentials for the virulence analysis of X. oryzae pv. oryzae in China. The 13 rice lines with a single gene were used further to establish a system of races classification of X. oryzae pv. oryzae in China. IR24 and IRBB10 were susceptible to the isolates with several exceptions, whereas IRBB5, IRBB7 and IRBB21 were resistant. Based on the interactions between the isolates of X. oryzae pv. oryzae and the 13 near-isogenic rice lines, six single-gene rice cultivars (IRBB5, IRBB13, IRBB3, IRBB14, IRBB2 and IR24) were chosen as differentials, and the 285 tested isolates were classified into nine races. The reaction patterns of the nine races in order were: RRRRRR, RRRRRS, RRRRSS, RRRSSS, RRSSSS, RSRRRS, RSSRRS, RSSSSS and SSSSSS. The race frequencies were 10.18%, 10.53%, 4.91%, 10.18%, 24.21%, 5.96%, 11.23%, 22.46% and 0.35% respectively. The virulence of representative strains of eight Philippine races on 13 rice lines with a single gene was determined and compared with the Chinese races. The frequency distributions of X. oryzae pv. oryzae races were primarily described for the different regions in China.

Age-dependent Grey Mould Susceptibility and Tissue-specific Defence Gene Activation of Grapevine Berry Skins after Infection by Botrytis cinerea

Abstract: The correlation between the degree of maturity of grapevine berries and their susceptibility to infection by the grey mould fungus Botrytis cinerea was studied. Artificial inoculation with B. cinerea conidia of detached berries from cultivars Riesling and Pinot noir revealed an increasing susceptibility during the last weeks of berry ripening. Wound inoculation resulted in increased lesion formation when compared with inoculation of non-wounded berry skins. Lesion development after non-wounding inoculation was stimulated by the addition of nutrients. Riesling berries were more readily infected than Pinot noir berries, indicating that the Riesling berry skin is more easily colonized by the grey mould fungus. Analysis of defence gene activation in the berry skin tissue revealed increased transcript levels of phenylalanine ammonium lyase and stilbene synthase after inoculation with B. cinerea conidia, while mRNA abundance of osmotin was similar in inoculated and non-inoculated tissue. Our data indicate that properties of the grape berry skin, including its ability for infection-induced defence gene activation, are important for the outcome of grey mould infections.

Occurrence of Toxic Hexadepsipeptides in Preharvest Maize Ear Rot Infected by Fusarium poae in Poland

Abstract: Twenty-seven preharvest maize ears affected by Fusarium poae rot (disease score 36-100%) were selected in 1998 and 1999 in Poland and examined for the occurrence of toxic hexadepsipeptides: beauvericin (BEA), enniatin A, enniatin B and enniatin B 1 The identification of F. poae was confirmed by sequence analysis of variable internal transcribed spacer regions and compared with NCBI gene bank DNA sequences. Chemical analyses were performed by HPLC-MS. In 27 ears infected by F. poae were detected: BEA (trace to 46 μg/g) in 18 samples, enniatin A (trace to 37 μg/g) in nine samples, enniatin B (trace to 47 μg/g) in 15 samples and enniatin B, (trace to 25 μg/g) in 12 samples. When 20 strains of F. poae isolated from these samples were cultured on rice, all produced BEA (1.9-75 μg/g), three enniatin A (1.8-2μg/g), 12 enniatin B (1.1-5.1 μg/g) and eight enniatin B 1 (1.2-5.2 μg/g). Occurrence and quantification of enniatin A, enniatin B and enniatin B 1 and their co-occurrence with BEA in maize kernels is reported for the first time.

A Rapid and Reliable Method for Monitoring the Sensitivity of Sclerotinia sclerotiorum to Boscalid

Abstract: Boscalid is a new fungicidal compound registered against Sclerotinia sclerotiorum in different crops in several European countries. For a sustainable use of this compound, the sensitivity of the target pathogen has to be observed. A mycelial growth test in tissue culture plates with liquid YBA medium was developed for a rapid and reliable sensitivity determination. Isolates never exposed to boscalid and field isolates from oilseed rape and beans, which had been isolated after boscalid market introduction and mainly from fields with boscalid treatments, were monitored for their sensitivity. All isolates were totally inhibited within 1.0 mg active ingredient/1. The frequency distribution of the ED 50 values was similar both for those isolates which had never been exposed to boscalid and for those isolates collected in 2006.

Identification of Barley mild mosaic virus Isolates in Germany Breaking rym5 Resistance

Abstract: In winter and early spring 2004 unequivocal mosaic symptoms were detected for the first time in Germany on six plants of the barley cv. Tokyo' carrying the resistance gene rym5. By serological and electron microscopic investigations Barley mild mosaic virus (BaMMV) was identified in all plants and could be re-transmitted to cv. 'Tokyo' as well as to additional cultivars carrying rym5. In contrast to this, genotypes carrying the resistance genes ryml + rym5, Rym2, rym4, rym7, rym9, rymll, rym12, rym13, Rym14 Hb , rym15 or Rym16 Hb turned out to be resistant. Furthermore, the BaMMV isolates were not transmissible to different dicotyledonous species. Sequence analyses in the VPg coding region of RNA1 revealed differences to the known sequence of the original BaMMV isolate (BaMMV-ASLI, AJ 242725) and also of a French pathotype (BaMMV-Sil, AJ 544267, AJ 544268) which is also able to overcome the resistance mediated by rym5. At least in one location a spread of the area infested by this new strain was observed in 2004/2005 and 2005/2006.

Nematophagous Fungi Associated with Root Galls of Rice Caused by Meloidogyne graminicola and its Control by Arthrobotrys dactyloides and Dactylaria brochopaga

Abstract: Root galls of rice caused by Meloidogyne graminicola were examined for natural colonization by nematophagous fungi from four fields with different nematode infestations. Old galls from severely infested fields had a higher frequency of Monacrosporium eudermatum and Stylopaga hadra than young galls. The frequency of Arthrohotrys oligospora, Arthrobotrys dactyloides, Dactylaria brochopaga and Monacrosporium gephyropagum was lower. A greater proportion (%) of root galls were colonized by nematophagous fungi in those fields in which rice roots had a greater root gall index. This indicated that disease severity supported the colonization of galls by nematophagous fungi. In vitro predacity tests of four fungi showed that A. dactyloides was most effective in capturing and killing J 2 of Mel. graminicola followed by D. brochopaga and Mon. eudermatum. Application of inocula of A. dactyloides and D. brochopaga in soil infested with Mel. graminicola, respectively, reduced the number of root galls by 86% and of females by 94%, and eggs and juveniles by 94%. The application of these fungi to soil increased plant growth: shoot length by 42.7% and 39.8%, root length by 45.5% and 48.9%, fresh weight of shoot by 59.9% and 56.7% and fresh weight of root by 20.3% and 25.1%, respectively, compared to these parameters for plants grown in nematode-infested soil.