Disease risk assessment of sugar beet root rot using quantitative real-time PCR analysis of <Emphasis Type="Italic">Aphanomyces cochlioides</Emphasis> in naturally infested soil samples

Sugar beet root rot, caused by the oomycete Aphanomyces cochlioides, is a serious and economically important disease of sugar beets world-wide. Today, disease risk assessment consists of a time-consuming greenhouse bioassay using bait plants. In the present study, a real-time quantitative PCR (qPCR) assay for determination of A. cochlioides DNA in field-infested soil samples was developed and validated using the standard bioassay. The qPCR assay proved to be species-specific and was optimized to give high amplification efficiency suitable for target copy quantification. A high correlation (R² > 0.98, p < 0.001) with pathogen inoculum density was shown, demonstrating the suitability for monitoring soil samples. The limit of detection (LOD) was evaluated in several different soil types and varied between 1 and 50 oospores/g soil, depending on clay content. Soils with a high LOD were characterised as having a low clay content and high content of sand. Varying levels of the A. cochlioides target sequence were detected in 20 of the 61 naturally infested soil samples. Discrepancies between the bioassay and the qPCR assay were found in soils from low- and medium-risk fields. However, the qPCR diagnostic assay provides a potentially valuable new tool in disease risk assessment, enabling sugar beet growers to identify high-risk fields.     

Description and molecular phylogeny of <Emphasis Type="Italic">Ditylenchus gilanicus</Emphasis> n. sp. (Nematoda: Anguinidae) from northern forests of Iran

Commercial areas containing Eucalyptus plantations have expanded in recent years due to increased demands for pulp, paper and bioenergy. One of the threats that can reduce Eucalyptus production is the eucalyptus rust disease caused by Austropuccinia psidii, a biotrophic fungus that affects a broad range of Myrtaceae. An accurate diagnosis tool for the early detection of rust disease could be useful in breeding programs for selection of resistant plants against rust, in phytosanitary purposes or in rust epidemics studies. The aim of the present work was to develop a SYBR Green-based quantitative real-time PCR (qPCR) assay for the early detection and quantification of A. psidii in Eucalyptus grandis leaves. Three sets of primers based on the A. psidii ribosomal DNA intergenic space region (IGS), beta-tubulin and elongation factor genes were designed and evaluated. The assays using the IGS primer set resulted in the highest detection efficiency, detecting a lower limit of 0.5 pg of A. psidii DNA. Under artificial inoculation in plants, A. psidii was detected immediately after pathogen inoculation until 240 h post-inoculation using qPCR. In field validation of the method, A. psidii was detected using qPCR in naturally infected leaves with or without rust symptoms. This easy and fast method can be used for an efficient detection of A. psidii in E. grandis leaves. The implications of this tool for rust studies are discussed below.     

TaqMan PCR assay for detection and quantification of <Emphasis Type="Italic">Stagonosporopsis tanaceti</Emphasis> in pyrethrum seed and seedlings

Pyrethrum seed has an important role in the transmission of Stagonosporopsis tanaceti, the cause of ray blight disease of pyrethrum. A TaqMan probe based polymerase chain reaction (PCR) assay was developed to quantify the level of S. tanaceti inocula in pyrethrum seed and seedlings. Primer pair (St_qF3, St_qR2) was designed based on the intergenic spacer (IGS) region of S. tanaceti, which produced a 125 bp amplicon specific to S. tanaceti. TaqMan PCR assay using St_qF3, St_qR2 and a probe St_qP was highly specific against the genomic DNA of S. tanaceti, but did not amplify DNA of 14 related Stagonosporopsis species or other foliar pathogens of pyrethrum. The sensitivity limit of this assay was measured using the cycle threshold (C_t) value which ranged from 17.59 for 10 nanograms (ng) to 36.34 for 100 femtograms (fg) genomic DNA of S. tanaceti. There was a significant negative correlation (r = ?0.999, P < 0.001) between the C_t value and the percent of S. tanaceti infected seed. In addition, this TaqMan PCR assay detected latent infection within seedlings. This assay could be applied to test commercial seed and seedlings before deciding on the appropriate management practices.     

Development of qPCR assays to monitor the ability of <Emphasis Type="Italic">Gliocladium catenulatum</Emphasis> J1446 to reduce the cereal pathogen <Emphasis Type="Italic">Fusarium graminearum</Emphasis> inoculum in soils

A qPCR approach was developed to specifically monitor in soils Fusarium graminearum, the main agent responsible for Fusarium Head Blight, and the biocontrol agent Gliocladium catenulatum J1446 (Prestop®). For both fungi, the amplification efficacy of standard curves obtained by mixing pure fungal DNA and soil background DNA was high (qPCR efficacy>96% with R²?>?0.97) with a linear range from 10^?3 ng to 10 ng/μL. Our qPCR method allowed quantifying down to 1 μg of F. graminearum and G. catenulatum J1446 mycelium per g of soil. The strong correlation observed between fungal biomass and quantified DNA (R²?=?0.9927 and 0.9356 for F. graminearum and G. catenulatum J1446, respectively) supported the use of the primers to monitor both fungi in soils. Under our experimental conditions, the ability of Prestop® to reduce F. graminearum growth was significantly higher in autoclaved soil compared to living soils, suggesting that there is an antagonistic effect of the soil microbial communities. In contrast, G. catenulatum J1446 growth was mostly not affected by the presence of F. graminearum and was able to persist in both autoclaved and living soils after 15 days of incubation. These results indicate that our qPCR approach may be used to assess the success of soil colonization by a biocontrol agent and its control efficacy by monitoring the dynamics of the BCA and the targeted pathogen in soil.     

<Emphasis Type="Italic">Bradyrhizobium</Emphasis> isolated from huanglongbing (HLB) affected citrus trees reacts positively with primers for <Emphasis Type="Italic">Candidatus</Emphasis> Liberibacter asiaticus

Bradyrhizobium sp., a slow-growing nitrogen-fixing symbiotic bacterium of legumes and common root endophyte of other plants, is closely related to Candidatus Liberibacter asiaticus (Las), the uncultured putative pathogen associated with citrus huanglongbing (HLB). In attempts to isolate Las on a low-nutrient medium that had been used for the isolation of several uncultured bacteria of the alpha subclass of proteobacteria, slow-growing Bradyrhizobium spp. were isolated and identified by sequencing of 16S rDNA. The individual isolates tested weakly positive (Ct = 31.2–36.0) with the USDA primers commonly used in qPCR assays for Las in foliar tissues. Direct DNA extracts from roots of HLB symptomatic trees that contained sequences of Bradyrhizobium sp. had Ct values ranging from 31.2 to 36.5; sequences of Las were not present in those samples. Potential cross-reaction between DNA of members of the Rhizobiales and sequences amplified by the Las primers were tested in silico with the Primer-BLAST tool in NCBI. Similar to Las, Bradyrhizobium generated predicted 16S rDNA amplicon sizes of 78–79 bp with the qPCR primers and of 1167-1172 bp with the conventional PCR primers. Bradyrhizobium sequences of 16S rDNA had 1–7 mismatches and only 1 mismatch at the 3′ end of qPCR and conventional PCR primers confirming potential cross-reactivity. As Bradyrhizobium is usually not found in foliage, the USDA qPCR primers can be safely used to check leaves for the presence of Las, but a threshold value of 31.0 is recommended for Las detection in roots. Other primers should be tested for potential cross-reaction with members of the Rhizobiales.     

Pathogenicity and Fungicide Sensitivity of <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> and <Emphasis Type="Italic">R. cerealis</Emphasis> Isolates

The Rhizoctonia solani species consists of multinucleate isolates that belong to anastomosis groups AG1–AG3 and differ in virulence and host affinity. R. cerealis is a binucleate species of anastomosis group AG-D which causes sharp eyespot, a common plant disease in Poland. Rhizoctonia spp. is a ubiquitous soil pathogen that poses a significant threat for global crop production due to the absence of effective crop protection products. The aim of this study was to determine the virulence of R. solani and R. cerealis isolates towards Beta vulgaris, Zea mays, Triticum spelta and T. aestivum seedlings, to confirm the presence of endopolygalacturonase genes pg1 and pg5 in the genomes of the tested isolates and to evaluate the tested isolates’ sensitivity to triazole, strobilurin, imidazole and carboxamide fungicides. All tested isolates infected B. vulgaris seedlings. but none of them were virulent against Z. mays plants. R. solani isolates AG4 PL and AG2-2IIIB PL were characterized by the highest virulence (average infestation score of 2.37 and 2.53 points on a scale of 0–3 points) against sugar beet seedlings. The prevalence of infections caused by most of the analysed isolates (in particular R. solani AG4 J—11.8, and R. cerealis RC2—0.78) was higher in spelt than in bread wheat. The virulence of the analysed isolates was not correlated with the presence of pg1 and pg5 genes. The efficacy of the tested fungicides in controlling Rhizoctonia spp. infections was estimated at 100% (propiconazole + cyproconazole), 98.8% (penthiopyrad), 95.4% (tebuconazole) and 78.3% (azoxystrobin).     

Visual detection of <Emphasis Type="Italic">Didymella bryoniae</Emphasis> in cucurbit seeds using a loop-mediated isothermal amplification assay

A method was developed using a Loop-mediated isothermal amplification assay (LAMP) for detecting Didymella bryoniae in cucurbit seeds. The LAMP primers were designed based on the DNA-dependent RNA polymerase II RPB140 gene (RPB2) from D. bryoniae. Calcein was used as an indicator for the endpoint visual detection of DNA amplification. The LAMP assay was conducted in isothermal (65 °C) conditions within 1 h. The detection threshold of the LAMP assay was 10 pg of genomic DNA and D. bryoniae was detected in 100 % of artificially infested seedlots with 0.05 % infestation or greater. With the LAMP assay, 16 of 60 watermelon and muskmelon seedlots collected from Xinjang province were determined to be positive for D. bryoniae. In contrast, a real-time PCR assay determined that 11 of the 60 seedlots from Xinjiang province were positive for the pathogen. These results showed that the LAMP technique was simple, rapid and well suited for detecting D. bryoniae DNA, especially in seed health testing.     

Effect of fumonisin B<Subscript>1</Subscript> on the emergence,growth and ceramide synthase gene expression of cowpea (<Emphasis Type="Italic">Vigna unguiculata</Emphasis> (L.) Walp)

Rice production is currently expanding from the south-eastern regions of Australia into northern Australia where indigenous species of wild rice occur widely. A survey of fungal diseases on wild (Oryza australiensis, Oryza spp.) and cultivated rice (Oryza sativa) in North Queensland, Australia, in May 2014 revealed a diverse range of fungal genera species, including important pathogens of cultivated rice. Whilst a single isolate of Magnaporthe oryzae (causal agent of rice blast) was obtained from wild rice, Bipolaris oryzae (causal agent of brown spot) was the predominant pathogen detected under North Queensland conditions. For the first time for Australia, we report Rhizoctonia oryzae-sativae (causal agent of aggregate sheath spot) occurring on wild rice. Other pathogens detected on wild rice included Curvularia lunata, Cochliobolus intermedius, Cochliobolus geniculatus, and Fusarium equiseti present in the majority of wild rice samples. Nearby cultivated rice fields harboured additional pathogens not found in wild rice including Fusarium graminearum, Leptosphaeria spegazzinii and Cochliobolus lunatus, causing scab disease, glume blight and leaf blight, respectively. We also confirmed that Bipolaris oryzae from wild rice can infect cultivated rice. This study highlights the importance of wild rice species as alternative hosts harbouring pathogens of cultivated rice and the likely disease threats to expansion of cultivated rice into the same region(s) where wild rice is endemic.     

Gene-based analysis of <Emphasis Type="Italic">Puccinia</Emphasis> species and development of PCR-based marker to detect <Emphasis Type="Italic">Puccinia striiformis</Emphasis> f. sp. <Emphasis Type="Italic">tritici</Emphasis> causing yellow rust of wheat

Stripe rust is considered as the current major rust disease affecting winter cereal production across the world. A quick, reliable PCR-based marker was developed here to detect, identify and rapidly monitor Puccinia striiformis f. sp. tritici (Pst) in wheat-growing areas. Three respective sets of primers, designed from β-tubulin, squalene monooxygenase and ketopantoate reductase genes selected from the full genome of Puccinia striiformis f. sp. tritici, amplified sequences of 239, 358 and 1518 bp, respectively, in Pst pathotypes. A fragment of 1518 bp unique to Pst pathotypes was amplified using primer set PstKeto F1_30/Pst KetoR1_1547 and distinguished the pathogen clearly from different Puccinia spp. and other fungal pathogens. The detection limit of the marker (KetoPstRA₁₅₀₀, accession no. KU240073) by conventional PCR assay was 10 pg. This marker could detect the pathogen in the host before symptom expression. The sensitivity and utility of the marker were further enhanced in a qPCR-based assay that was developed with a newly designed primer set PstKeto F1_1246/Pst KetoR1_1547, which amplified a product of 302 bp and detected as little as 10 fg of DNA. This PCR/qPCR based marker is suitable for studying cultivar resistance, which requires accurate quantification of the pathogen in diseased host tissue.     

Increased susceptibility of potato to <Emphasis Type="Italic">Rhizoctonia</Emphasis> diseases in <Emphasis Type="Italic">Potato leafroll virus</Emphasis>-infected plants

In Hokkaido potato fields, tubers produced from the plants with leaf curl symptoms caused by potato leaf roll virus (PLRV) were noted to be more densely covered with Rhizoctonia sclerotia. This observation led us to hypothesize that potato infected with PLRV would have an increased susceptibility to Rhizoctonia solani. To test this hypothesis, in a pot experiment, we inoculated PLRV-infected mother tubers with Rhizoctonia. As a result, PLRV-infected plants produced significantly fewer and smaller tubers than virus-free plants did, suggesting that PLRV-infected plants are more susceptible than virus-free plants to R. solani. Virus-free seed tubers should thus be used to reduce Rhizoctonia diseases.     

Development of a TaqMan probe-based insulated isothermal PCR (TiiPCR) for the detection of <Emphasis Type="Italic">Acidovorax citrulli</Emphasis>, the bacterial pathogen of watermelon fruit blotch

Watermelon (Citrullus lanatus) is an important crop of the Cucurbitaceae family in fruit production worldwide. During its production, bacterial fruit blotch (BFB) caused by Acidovorax citrulli (Acidovorax avenae subsp. citrulli) is an important limiting factor on the volume and value of crops. This pathogen is known as a seed-borne pathogen, and the infested seeds can be a primary source of inoculum. Hence, a rapid and sensitive method for detecting A. citrulli on seeds would be an important tool in the management of BFB. In this study, we sought to develop a method to detect A. citrulli bacterial cells based on a TaqMan probe-based insulated isothermal PCR (TiiPCR) assay. Firstly, the specific primers and probe were designed based on a specific DNA fragment from the genome of A. citrulli. Then, PCR amplification was performed with the plasmid DNA to adjust the components of the PCR reagents, such as the concentrations of primers, magnesium chloride, and Taq DNA polymerase. Results revealed that 10 copies of plasmid DNA were detectable within the modified reagents by TiiPCR. Moreover, 10 bacterial cells in each reaction tube were detectable at a 100 % detection rate in this condition with a fluorescent signal intensification over 1.8. Based on these results, we concluded that a specific, rapid, and sensitive method based on TiiPCR had been successfully developed to detect bacterial cells of A. citrulli.     

Trichothecene genotype and genetic variability of <Emphasis Type="Italic">Fusarium graminearum</Emphasis> and <Emphasis Type="Italic">F. cerealis</Emphasis> isolated from durum wheat in Argentina

Fusarium head blight (FHB) is one of the most important fungal diseases affecting wheat worldwide and it is caused mainly by species within the Fusarium graminearum species complex (FGSC). This study evaluated the presence of FGSC in durum wheat from the main growing area in Argentina and analyzed the trichothecene genotype and chemotype of the strains isolated. Also, the genetic variability of the strains was assayed using ISSR markers. Molecular analysis revealed that among the strains isolated and identified morphologically as F. graminearum, there were 14 strains identified as F. cerealis. Also, it revealed that durum wheat grains were mostly contaminated by F. graminearum, being this the only species reported so far, within the FGSC, affecting durum wheat in Argentina. Analysis of molecular variance (AMOVA) indicated a high genetic variability within rather than between F. graminearum populations. All F. graminearum strains presented 15ADON genotype and were able to produce DON while all F. cerealis strains presented the NIV genotype and most of them were able to produce this toxin. The finding of F. cerealis in durum wheat grains indicates the need for investigating if this fungus is the responsible for the NIV contamination found in wheat in Argentina.     

The role of <Emphasis Type="Italic">Ustilaginoidea virens</Emphasis> sclerotia in increasing incidence of rice false smut disease in the subtropical zone in China

Rice false smut is heavily and increasingly occurring in subtropical zones in China in the past decades. The pathogen of the disease, Ustilaginoidea virens, can produce both chlamydospores and sclerotia, and the sclerotia seem to form frequently in temperate or high-altitude regions in China. Which of these structures play a dominant role in the pathogen’s life cycle in subtropical zones remains unclear. Here we found that Ustilaginoidea virens could produce a great number of sclerotia in subtropical zones and the maximal number of sclerotia could reach to 2.25 million per hectare. In the year with relatively low autumn temperatures, the disease severity and sclerotia numbers of U. virens increased significantly. Although there was a few sclerotia in subtropical zones capable of overwintering successfully, one individual sclerotium could produce large numbers of ascospores. In the rice-growing paddy field, the ascospores could be trapped in both temperate and subtropical zones in May–September, when rice was at the booting stage, the critical infection period of rice false smut. This suggested that the sclerotia of rice false smut in subtropical zone played an important role in the life cycle of Ustilaginoidea virens and acted as the primary inoculum. Experiments in the laboratory showed that mature sclerotia of rice false smut remained dormant for about 2–5 months, and that light was essential for fruiting body differentiation. As with ergot, the fruiting bodies of Ustilaginoidea virens secreted sticky droplets on the stromata that prevented the ascospores from dispersing into the air, implying that the transfer of ascospores of Ustilaginoidea virens to rice plants in paddy field needed an intermediary vector.     

Assessment of latent infection with <Emphasis Type="Italic">Verticillium longisporum</Emphasis> in field-grown oilseed rape by qPCR

Improvement of cultivar resistance is the key strategy to control the host-specialized pathogen Verticillium longisporum in oilseed rape (OSR). A special feature of this pathogen is its systemic, non-homogenous and delayed colonization of the plant xylem resulting in an extended symptomless period of latency. As a result, severity of infection in the field is difficult to score as it becomes apparent only at crop maturity stages when it may be confused with natural senescence. Assessment of Verticillium disease severity in OSR by visual scoring of microsclerotia on harvested stubbles unsatisfactorily reflects genotypic resistance as it is strongly affected by the ripening stage of the plant. To overcome these limitations, we developed a qPCR method, which unambiguously differentiates levels of quantitative resistance to V. longisporum in OSR genotypes under field conditions. The specificity and sensitivity of two primer pairs targeting ITS or tubulin loci in the V. longisporum genome were tested. While tubulin primers showed a high specificity to V. longisporum isolates, ITS primers exhibited a significantly higher sensitivity in detecting fungal DNA in stem tissue (limit of quantification =0.56 fg DNA) of field-grown pre-symptomatic plants. The best discrimination of resistant and susceptible OSR cultivars based on fungal DNA analysis in stem tissue was achieved at growth stage 80, at the transition of fungal vascular growth in viable plants to saprotrophic colonization of senescent stem tissues. Field screening data obtained with qPCR at growth stage 80 confirmed results from greenhouse testing thus corroborating the relevance and reliability of seedling assays for determining cultivar responses to V. longisporum in the field, as a useful tool for breeders in first selection of elite OSR genotypes with improved resistance to Verticillium.     

Mechanism of in vitro antagonism of phytopathogenic <Emphasis Type="Italic">Scelrotium rolfsii</Emphasis> by actinomycetes

Sclerotium rolfsii (Sr), a soil-borne fungal pathogen, causes disease in a wide range of crops. Recently, we identified five actinomycetes (Streptomyces globisporus subsp. globisporus, S. globisporus, S. flavotricini, S. pactum, and S. senoensis) showing significant inhibitory effects on plant pathogens. In this study, the effects of the five actinomycetes for the biocontrol of Sr were investigated using the plate culture method and microscopy examination. Two actinomycetes with higher inhibitory effect were subsequently examined for the inhibition of sclerotial germination of Sr in unsterile soil in vitro. The cell-free cultures of five actinomycetes mediated significant inhibition of hyphal growth and sclerotial formation and germination of Sr. All actinomycete strains exhibited the ability to produce extracellular cell wall degrading enzymes in the culture conditions. The crude enzyme suspensions of S. flavotricini and S. pactum hydrolyzed the cell wall of Sr. At a dose of 1 g per kilogram soil, the solid formulations of S. flavotricini and S. senoensis prevented germination of 24% and 68% of sclerotia, respectively. Our results provide evidence of effective strains for the biocontrol of Sr, in addition to a further understanding of the underlying mechanism.     

In vitro competition between <Emphasis Type="Italic">Fusarium graminearum</Emphasis> and <Emphasis Type="Italic">Epicoccum nigrum</Emphasis> on media and wheat grains

Competitive effects between Fusarium graminearum, causing Fusarium head blight, and the endophyte Epicoccum nigrum, were performed in in vitro competition assays between the two species. Two E. nigrum isolates were isolated from wheat grains and tested as competitors against two F. graminearum isolates. A dual petri dish assay showed that E. nigrum reduced the mycelial growth of F. graminearum and vice versa. A glass slide assay revealed that E. nigrum crude cultural filtrate also had reducing effect on the growth of F. graminearum comparable to that of E. nigrum spore suspensions. Microscopy showed hyphae of F. graminearum and E. nigrum with many side branches when in close proximity, in contrast to pronounced apical hyphal growth when growing alone. Combinations of F. graminearum and E. nigrum on sterilised wheat grains were studied over time by qPCR. F. graminearum biomass was significantly reduced in inoculations applying E. nigrum three days prior to F. graminearum. In conclusion, these results showed competition and mycelial behaviour effects between F. graminearum and E. nigrum and support that E. nigrum may have potential to reduce F. graminearum infections in wheat. Competition experiments should be carried out in planta to study the interaction further.     

Morphological and molecular variability among Indian isolates of <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> causing banded leaf and sheath blight in maize

Rhizoctonia solani, a devastating soil borne fungus inciting banded leaf and sheath blight (BLSB) disease is a constraint in maize production and improvement program. Rhizoctonia isolates collected from seven diverse maize cropping zones of India were examined for morphological and molecular variability. All the tested isolates caused symptoms of BLSB on maize and were also cross infective on rice and sugarcane hosts, but showed significant variability in hyphal diameter, mean hyphal cell size, weight, size and distribution of scleorotia, culture pigmentation, incubation period, pathogenicity and expression of symptoms. Neighbour joining cluster analysis placed the 62 isolates of R. solani into four major groups, A, B, C and D. Group A was more diverse and included isolates of diverse agro-ecological zones. The cluster analysis corresponded well with principle component analysis. Pathogenicity testing of R. solani isolates on maize genotype (CM 501) revealed highly variable virulence pattern of the pathogen population suggesting its high evolutionary potential, and hence adaptability to diverse geographical regions. The study reveals a strong evidence of inherent potential of the R. solani isolates to survive in diverse ecological zones and its probable spread to other maize cultivars across India. Sequence comparisons of the internal transcribed sequence-ribosomal DNA region of 62 isolates did not reveal much diversity among the isolates. Majority of the isolates (n?=?61) clustered together with anastomosis group (AG) AG1-IA used as reference strain in the phylogram, distinct from AG1-IB, AG2–2IIIB and Waitea circinata used as reference strains. BLSB isolates representing distinct geographical locations shared identical sequences indicating long-distance dispersal of the pathogen. The study confirms that the genetic flexibility of the pathogen allows for its adaptation to variable ecological niches and long-distance introduction of new genotypes into the region. The study emphasizes that epidemiological studies may complement the molecular studies.     

Histological observation of cucumber infected with <Emphasis Type="Italic">Corynespora cassiicola</Emphasis>

Erwinia amylovora is the causative agent of fire blight, which is a destructive bacterial disease of rosaceous plants. In Hungary Erwinia amylovora (Burrill) Winslow et al. was first detected in 1996. Since the appearance of fire blight, E. amylovora samples have been collected from different host plats from various geographic locations. A motif of eight nucleotides (ATTACAGA) is repeated 3–15 times in the PstI fragment of the pEa29 plasmid in Erwinia amylovora strains, and represents a valuable tool for strain classification. The number of short-sequence DNA repeats in plasmid pEa29 of 30 Hungarian isolates were determined by PCR assays and they ranged from five to ten. The SSR test is suitable for distinguishing the individual strains between the E. amylovora isolates. The examined isolates showed high pathogenicity on immature pear fruits. Several biochemical techniques, such as miniaturized API 20E, were applied on the samples. Differences were also revealed in microbiological assays like levan formation and colony morphology on semi-selective media. Examining the Hungarian Erwinia amylovora population by molecular analysis we can draw the conclusion that the population consists of different strains, which shows great diversity. E. amylovora is a widespread pathogen in Hungary, which is supported by the 30 strains isolated from various host plants from many parts of the country. The phenotypic diversity-evaluation of the E. amylovora strains showed, that they differ metabolically, like other plant pathogenic bacteria as reported by several authors. This is the first report on the diversity of E. amylovora strains isolated from Hungary.     

Exploring host-pathogen combinations for compatible and incompatible reactions in grapevine downy mildew

Rhizoctonia leguminicola, the traditional name for the causal agent of blackpatch of red clover (Trifolium pratense) and other legumes, produces alkaloids, one of which causes livestock to salivate excessively. Fungal presence is generally confirmed by microscopy, disappearance of symptoms in livestock after removal of suspect forage, and chromatography of the alkaloid slaframine, in legume tissue. Use of the polymerase chain reaction (PCR) to amplify a pathogen-specific DNA fragment would complement the other methods of pathogen identification. Primers were designed to the R. leguminicola ITS region, sequence provided by another laboratory. Two separate primer pairs each amplified a different fragment–one ~250 bp long (expected length 249 bp), and the other 300 to 400 bp long (expected length 368 bp)–in DNA extracted from cultures of R. leguminicola. Under the experimental conditions, the primers to the larger fragment amplified a stronger band, and a minimum of 0.1 ng DNA per reaction was needed to produce a detectable band. With the primers to the 368-bp fragment, a band 300 to 400 bp long was also amplified in DNA extracted from red clover (cultivar Kenland) inoculated with R. leguminicola and harvested 70 h post inoculation. No amplification with this primer set occurred in DNA extracted from mock-inoculated red clover plants, supporting the likelihood that the primers amplified R. leguminicola DNA extracted from inoculated red clover. This primer set did not amplify DNA extracted from a red clover isolate of the legume pathogen Stemphylium sarcinaeforme, or DNA extracted from two isolates of the legume pathogen Colletotrichum trifolii, indicating specificity for R. leguminicola DNA. Lack of amplification of alfalfa DNA indicated that the R. leguminicola primers will be useful for testing for the presence of blackpatch in alfalfa.     

An environmentally sustainable approach for the management of <Emphasis Type="Italic">Phaeoacremonium minimum</Emphasis>, the main agent of wood diseases in <Emphasis Type="Italic">Actinidia deliciosa</Emphasis>

Among the fungi associated with the kiwi wood diseases, the vascular pathogen Phaeoacremonium minimum can infect plants already at nursery stage, without any external symptoms. At the moment, there are not effective control strategies. The effect of soil treatments applicable in organic agriculture was evaluated in two-years experiments on potted kiwi plants artificially inoculated with P. minimum. The soil treatments were based on commercial formulations of iron chelate, silicon, neem paste, Trichoderma afroharzianum strain T22, and cover cropping with selected perennial Poaceae. Cover cropping and iron chelate treatments enhanced the iron availability for the plants and significantly reduced wood necrosis caused by the pathogen. Both treatments also produced an increase of hairy root proliferation, so the plants were able to better cope with stress conditions. Laboratory assays showed the role of iron on the pathogen growth and its pathogenesis enzyme activities.