Verticillium Spp Research Articles

Rapid and Sensitive Detection of Verticillium dahliae from Soil Using LAMP-CRISPR/Cas12a Technology.

Cotton Verticillium wilt is mainly caused by the fungus Verticillium dahliae, which threatens the production of cotton. Its pathogen can survive in the soil for several years in the form of microsclerotia, making it a destructive soil-borne disease. The accurate, sensitive, and rapid detection of V. dahliae from complex soil samples is of great significance for the early warning and management of cotton Verticillium wilt. In this study, we combined the loop-mediated isothermal amplification (LAMP) with CRISPR/Cas12a technology to develop an accurate, sensitive, and rapid detection method for V. dahliae. Initially, LAMP primers and CRISPR RNA (crRNA) were designed based on a specific DNA sequence of V. dahliae, which was validated using several closely related Verticillium spp. The lower detection limit of the LAMP-CRISPR/Cas12a combined with the fluorescent visualization detection system is approximately ~10 fg/μL genomic DNA per reaction. When combined with crude DNA-extraction methods, it is possible to detect as few as two microsclerotia per gram of soil, with the total detection process taking less than 90 min. Furthermore, to improve the method's user and field friendliness, the field detection results were visualized using lateral flow strips (LFS). The LAMP-CRISPR/Cas12a-LFS system has a lower detection limit of ~1 fg/μL genomic DNA of the V. dahliae, and when combined with the field crude DNA-extraction method, it can detect as few as six microsclerotia per gram of soil, with the total detection process taking less than 2 h. In summary, this study expands the application of LAMP-CRISPR/Cas12a nucleic acid detection in V. dahliae and will contribute to the development of field-deployable diagnostic productions.

Isolation and Identification of Fungi Associated with Fruit Rot Disease of Tomato (Solanum lycopersicum L.) in the Southern Guinea Savannah, Nigeria

Rot disease is a major threat to tomato fruit production and postharvest handling in major tomato growing areas in Nigeria. Rotted tomato fruits were randomly collected from farmers’ farms for the purpose of isolation and identification of rot causing fungi in Tarka, Benue State, Nigeria between July and December, in 2015 and 2016. Results revealed that Aspergillus flavus, A. niger, Alternaria solani, Phytophthora infestans, Oidium neolycopersici, Sclerotium rolfsii, Fusarium oxysporum, Verticillium spp and Colletotrichum spp caused tomato fruit rot in the area. In both years, the lowest number of fungi was recorded in the months of July and December while the highest number was obtained in September. The mean number of fungi isolated showed that S. rolfsii was the least with 3.17 in 2015 and 1.66 in 2016 while F. oxysporum was the highest with 14.33 in 2015 and 9.33 in 2016. There were more fungi isolated in 2015 than in 2016. There were significant differences (P≤0.05) in the mean number of A. niger, P. infestans, O. neolycopersici and F. oxysporum isolated in both years. In conclusion, farmers should handle tomato fruits with care, to reduce postharvest injury and damage in order to ensure the availability of disease free fruits and to increase the output for the global population.

Rapid and Sensitive Detection of Verticillium dahliae from Complex Samples Using CRISPR/Cas12a Technology Combined with RPA.

Verticillium wilt is primarily caused by the fungus Verticillium dahliae and represents one of the most important worldwide soilborne plant diseases. The causal agent can be spread by microsclerotia and conidia attached to seeds during national/international trade or in soil between fields. Consequently, accurate, sensitive, and rapid detection of V. dahliae from complex samples is critical for restricting entry of the pathogen to a new region/environment and enforcing early management of Verticillium wilt. Based on CRISPR/Cas12a and recombinant polymerase amplification (RPA) technologies, we developed an accurate, sensitive, and rapid detection method for V. dahliae with paper-based lateral flow strips. A highly efficient and specific CRISPR RNA (crRNA) was designed for the GAPDH gene of V. dahliae and was validated using several closely related Verticillium spp. Excluding the time required for the DNA extraction from the complex samples, a minimum of 40 min was required for the detection time. The RPA-CRISPR/Cas12a detection system had a lower detection limit of ∼10 copies of genomic DNA per reaction and was able to successfully detect as little as one microsclerotium per gram of soil. In addition, field samples displaying symptoms suggestive of V. dahliae were able to be positively identified for the presence of V. dahliae. Taken together, this study broadens the applications of CRISPR/Cas12a nucleic acid detection to soilborne crop diseases and will contribute to the future development of field-deployable diagnostic tools.

Non-target effects of Verticillium nonalfalfae isolate Vert56 used for biological control of Ailanthus altissima on agricultural crops known to be generally susceptible to Verticillium spp.

Tree of Heaven (Ailanthus altissima), a tree species native to China, was introduced into Europe as an ornamental plant about 250 years ago. It is known for its invasive spread with negative impacts on natural ecosystems as well as human infrastructure. Its control is inevitable in some areas, e.g. transportation corridors, for safety reasons. Furthermore, management actions are mandatory according to EU legislation, but common strategies are time-consuming, costly, only moderately effective and, in case of herbicide treatments, controversially discussed due to environmental issues. Isolate Vert56 of the vascular wilt fungus Verticillium nonalfalfae has been proven in previous studies to be highly adapted and highly virulent to Ailanthus, while inoculation of potentially susceptible tree species with this isolate showed no evidence of negative non-target effects.This study focused on non-target effects of isolate Vert56 on agricultural crops known to be generally susceptible to Verticillium spp. In a greenhouse experiment, seedlings of seven different species (including Ailanthus as a positive control) were artificially inoculated via root dipping or, in the case of seedlings of two potato varieties, by direct addition of the inoculum into the pot.All plants were subsequently assessed for symptoms during the following two months. After assessment, plants were harvested to determine dry biomass. Likewise, tissue samples were used for re-isolation of the pathogen. Besides morphological identification, 25% of the obtained fungal cultures were analyzed with species-specific primers. The pathogen was consistently detected in all species. In addition to Ailanthus, three species (Cucumis sativus, Petunia × hybrid, Spinacia oleracea) developed severe wilting symptoms and showed a decrease in biomass compared to control plants. One species, Solanum lycopersicum, displayed a decrease in biomass although only slight wilting symptoms were observed within a short period after inoculation. Humulus lupulus, Medicago sativa and Solanum tuberosum were not negatively affected by V.nonalfalfae isolate Vert56. These results indicate that safety precautions must be taken, or at least considered, when growing susceptible plants in the vicinity of Vert56-treated Ailanthus, but they also show that this biological control agent is suitable for the treatment of Ailanthus on/next to agricultural land.

English

Tomato crop is affected with several diseases that lead to decreased yield every year. Among those diseases, tomato wilt is the most significant disease caused by soil borne pathogens i.e. Fusarium solani, Fusarium oxysporum, and Verticillium spp. In the present study, survey was conducted in tomato growing areas of Punjab. Pathogens associated with plants were Fusarium oxysporum, Alternaria solani, parasitic nematodes, Pythium, Verticillium dahliae and mosaic virus. The survey was conducted at three different stages of plant development and disease incidence and severity were correlated with environmental factors. This survey was conducted for consecutive two years (2017-18:2018-19). It was shown that fusarium wilt can infect all growing stages (nursery, flowering and fruiting) of tomato plant. Moreover, high temperature and high humidity favored disease prevalence. Huge damage was witnessed due to high temperature and relative humidity up to a certain limit. Furthermore, it was also observed that alternaria blight, verticillium wilt, nematodes and mosaic virus also attacked at different growth stages to different extents along with fusarium wilt. Plants were more vulnerable at fruiting stage to all diseases. The present study will be helpful for devising a proper management strategy for fusarium wilt disease in tomato crop.

Pseudomonas Strains Induce Transcriptional and Morphological Changes and Reduce Root Colonization of Verticillium spp.

Phytopathogenic Verticillia cause Verticillium wilt on numerous economically important crops. Plant infection begins at the roots, where the fungus is confronted with rhizosphere inhabiting bacteria. The effects of different fluorescent pseudomonads, including some known biocontrol agents of other plant pathogens, on fungal growth of the haploid Verticillium dahliae and/or the amphidiploid Verticillium longisporum were compared on pectin-rich medium, in microfluidic interaction channels, allowing visualization of single hyphae, or on Arabidopsis thaliana roots. We found that the potential for formation of bacterial lipopeptide syringomycin resulted in stronger growth reduction effects on saprophytic Aspergillus nidulans compared to Verticillium spp. A more detailed analyses on bacterial-fungal co-cultivation in narrow interaction channels of microfluidic devices revealed that the strongest inhibitory potential was found for Pseudomonas protegens CHA0, with its inhibitory potential depending on the presence of the GacS/GacA system controlling several bacterial metabolites. Hyphal tip polarity was altered when V. longisporum was confronted with pseudomonads in narrow interaction channels, resulting in a curly morphology instead of straight hyphal tip growth. These results support the hypothesis that the fungus attempts to evade the bacterial confrontation. Alterations due to co-cultivation with bacteria could not only be observed in fungal morphology but also in fungal transcriptome. P. protegens CHA0 alters transcriptional profiles of V. longisporum during 2 h liquid media co-cultivation in pectin-rich medium. Genes required for degradation of and growth on the carbon source pectin were down-regulated, whereas transcripts involved in redox processes were up-regulated. Thus, the secondary metabolite mediated effect of Pseudomonas isolates on Verticillium species results in a complex transcriptional response, leading to decreased growth with precautions for self-protection combined with the initiation of a change in fungal growth direction. This interplay of bacterial effects on the pathogen can be beneficial to protect plants from infection, as shown with A. thaliana root experiments. Treatment of the roots with bacteria prior to infection with V. dahliae resulted in a significant reduction of fungal root colonization. Taken together we demonstrate how pseudomonads interfere with the growth of Verticillium spp. and show that these bacteria could serve in plant protection.

Searching for New Beneficial Bacterial Isolates of Wild Raspberries for Biocontrol of Phytopathogens-Antagonistic Properties and Functional Characterization.

The threat caused by plants fungal and fungal-like pathogens is a serious problem in the organic farming of soft fruits. The European Commission regulations prohibit some commercially available chemical plant protection products, and instead recommend the use of natural methods for improving the microbial soil status and thus increasing resistance to biotic stresses caused by phytopathogens. The solution to this problem may be biopreparations based on, e.g., bacteria, especially those isolated from native local environments. To select proper bacterial candidates for biopreparation, research was provided to preliminarily ensure that those isolates are able not only to inhibit the growth of pathogens, but also to be metabolically effective. In the presented research sixty-five isolates were acquired and identified. Potentially pathogenic isolates were excluded from further research, and beneficial bacterial isolates were tested against the following plant pathogens: Botrytis spp., Colletotrichum spp., Phytophthora spp., and Verticillium spp. The eight most effective antagonists belonging to Arthrobacter, Bacillus, Pseudomonas, and Rhodococcus genera were subjected to metabolic and enzymatic analyses and a resistance to chemical stress survey, indicating to their potential as components of biopreparations for agroecology.

Mycorrhizal Fungi as Bioprotectors of Crops Against Verticillium Wilt-A Hypothetical Scenario Under Changing Environmental Conditions.

The association that many crops can establish with the arbuscular mycorrhizal fungi (AMF) present in soils can enhance the resistance of the host plants against several pathogens, including Verticillium spp. The increased resistance of mycorrhizal plants is mainly due to the improved nutritional and water status of crops and to enhanced antioxidant metabolism and/or increased production of secondary metabolites in the plant tissues. However, the effectiveness of AMF in protecting their host plants against Verticillium spp. may vary depending on the environmental factors. Some environmental factors, such as the concentration of carbon dioxide in the atmosphere, the availability of soil water and the air and soil temperatures, are predicted to change drastically by the end of the century. The present paper discusses to what extent the climate change may influence the role of AMF in protecting crops against Verticillium-induced wilt, taking into account the current knowledge about the direct and indirect effects that the changing environment can exert on AMF communities in soils and on the symbiosis between crops and AMF, as well as on the development, incidence and impact of diseases caused by soil-borne pathogens.

Effect of chemical alternatives to methyl bromide on soil-borne disease incidence and fungal populations in Spanish strawberry nurseries: A long-term study.

Chloropicrin (PIC) mixtures of 1,3-dichloropropene and chloropicrin (DD:PIC), dazomet, and metam sodium (MS) have been applied as chemical alternatives to methyl bromide (MB) in Spanish strawberry nurseries since MB was banned as a soil fumigant in 2005. These chemical alternatives were applied to soil in two Spanish strawberry nurseries between 2003 and 2017 to test their efficacy against the main crown and root disease and soil fungal populations in comparison with the use of MB and PIC (MB:PIC). These chemicals were applied at several doses with different application methods under plastic films. Crown and root disease incidence was calculated as the percentage of plants with symptoms caused by soil-borne pathogens. Soil fungal populations were estimated as colony forming units per gram of dry soil. All chemicals significantly reduced soil-borne fungal disease incidence and fungal population in both nurseries over the years. Phytophthora cactorum and Fusarium spp. were the main pathogens causing soil-borne diseases, followed by Verticillium spp. MB:PIC remained the treatment that best controlled P. cactorum. MS and DD:PIC controlled Fusarium disease to a lesser extent than MB:PIC and dazomet in both nurseries. MB:PIC and PIC were the two treatments that most reduced Verticillium spp. The population of Verticillium spp. declined and the presence of other species such as Colletotrichum spp. and Rhizoctonia spp. was minimal during the study. Chemicals are necessary to obtain healthy strawberry plants. The use of chemical alternatives to MB has resulted in changes in the incidence of soil-borne diseases and soil fungal populations in strawberry nurseries. Dazomet was an effective alternative to MB as a soil-borne disease control, except against Verticillium spp. MB alternatives in strawberry nursery soils have caused Fusarium spp. to displace Verticillium spp.

Antalya ilinde yetiştirilen çileklerde hastalığa neden olan fungal etmenler

Bu çalışmada, Antalya ilindeki çilek ekim alanlarında hastalığa neden olan fungal patojenlerin belirlenmesi amacıyla, ilde çilek yetiştiriciliğinin yapıldığı 8 ilçede, toplamda 28 farklı serada örnekleme yapılarak, toprak ve hastalıklı bitki örnekleri alınmıştır. Örneklerden yapılan izolasyonlar sonucunda 12 fungus cinsi belirlenmiştir. Topraktan yapılan izolasyonlarda %96.43 ile yaygınlık oranı en yüksek fungus Rhizoctonia solani olurken, %75 ile Fusarium spp. ve %35.71 ile Pythium sp. onu izlemiştir. Gliocladium roseum, Alternaria alternata, Aspergillus niger, Penicillium sp., Rhizopus stolonifer, Botrytis cinerea ve Verticillium sp. ise toprak örneklerinde düşük yaygınlık oranlarıyla bulunan diğer funguslar olmuştur. Yaprak örneklerinde Alternaria alternata %96.43 oranıyla en yaygın fungus olarak belirlenirken, %71.43 oranıyla Mycosphaerella fragariae onu izlemiş, Cladosporium cladosporioides’in yaygınlık oranı ise %3.57 olmuştur. Meyvelerden yapılan izolasyonlarda ise %85.71 oranıyla Botrytis cinerea en yaygın fungus olarak saptanmış, %35.71 ile Penicillium sp. ve %10.71 ile Aspergillus niger çürük meyvelerden izole edilen diğer funguslar olmuştur. Laboratuvar koşullarında yürütülen patojenite denemelerinde tüm funguslar kendilerine özgü belirtileri oluşturmuşlardır. Bu sonuçlara göre; Antalya ilinde yetiştirilen çileklerde A. alternata, M. fragariae ve B. cinerea en önemli patojenler olarak belirlenirken, R. solani, Fusarium spp., Pythium spp., Verticillium spp. gibi bazı toprak kökenli patojenlerin de ciddi kayıplara neden olabileceği ortaya konulmuştur.

Effects of phosphite on the in vitro growth of Verticillium nonalfalfae and Verticillium dahliae and on their in vivo ability to infect potato plants

Verticillium spp. are soil-borne fungi containing many pathogenic species that affect a wide range of cultivated plants. In potatoes, Verticillium dahliae, V. nonalfalfae and V. albo-atrum cause Verticillium wilt or, together with other pathogens, are responsible for the occurrence of the potato early dying disease. Verticillium pathogens are notoriously difficult to control, and techniques proved to be efficient, such as chemical soil fumigation, are causing environmental concerns worldwide. Phosphites (Phi) represent a class of chemicals with a good environmental footprint, that are currently used to control oomycete pathogens, such as the potato late blight agent, Phytophtora infestans. To determine the potential inhibitory effects of Phi on Verticillium spp., isolates of V. dahliae and V. nonalfalfae were tested in vitro and the IC50 of Phi was found to vary between 60.9 and 481.9 μg/mL. The effects of Phi were further tested in field trials, carried for two years, using Phi-treated potato plants infected with a V. nonalfalfae isolate. Infection progression was assessed in both years by qPCR and was found to be significantly reduced in Phi-treated plants. The concentration of Phi in these plants, determined by high performance ion chromatography, was found to vary between 25.0 and 86.2 μg/mL in leaves and between 55.7 and 113.8 μg/mL in tubers. Even though the concentrations of Phi required to control Verticillium spp. were found to be relatively high compared to those needed to inhibit the development of oomycete pathogens, the results from this study indicate that Phi can limit the development of Verticillium spp.

Patógenos comunes de semilla de ajo de Aguascalientes y Zacatecas, México

El ajo es el cultivo hortícola de mayor importancia durante el invierno en los estados de Aguascalientes y Zacatecas, México. Se desconoce la presencia de daño abiótico y biótico en la semilla de ajo de ésta región, consecuentemente, el objetivo de éste trabajo fue determinar la incidencia de patógenos y daño abiótico en los bulbillos de ajo utilizados como semilla durante el año 2015. Se colectaron bulbos de ajo de diferentes variedades, los dientes de cada bulbo fueron separados y se contó el número de dientes sanos y dañados para su observación. Los bulbos de tres variedades de ajo (Chileno, Hermosillo y Plateado) no mostraron ningún tipo de daño. El patógeno más común fue el hongo Ulocladium spp. aunque otros como Alternaria spp., Fusarium spp., Curvularia spp, Rhizoctonia spp., Penicillium spp., and Verticillium spp., también fueron detectados. El ácaro Rhyzoglyphus fue detectado en solo cinco de las variedades colectadas. Daño químico fue detectado afectando 52% de las variedades colectadas. Las pérdidas de peso provocadas por daño abiótico o biótico fueron más severas en las variedades Durango, Calerense y Español.
 

Transfer of tomato immune receptor Ve1 confers Ave1-dependent Verticillium resistance in tobacco and cotton.

SummaryVerticillium wilts caused by soilborne fungal species of the Verticillium genus are economically important plant diseases that affect a wide range of host plants and are notoriously difficult to combat. Perception of pathogen(‐induced) ligands by plant immune receptors is a key component of plant innate immunity. In tomato, race‐specific resistance to Verticillium wilt is governed by the cell surface‐localized immune receptor Ve1 through recognition of the effector protein Ave1 that is secreted by race 1 strains of Verticillium spp. It was previously demonstrated that transgenic expression of tomato Ve1 in the model plant Arabidopsis thaliana leads to Verticillium wilt resistance. Here, we investigated whether tomato Ve1 can confer Verticillium resistance when expressed in the crop species tobacco (Nicotiana tabcum) and cotton (Gossypium hirsutum). We show that transgenic tobacco and cotton plants constitutively expressing tomato Ve1 exhibit enhanced resistance against Verticillium wilt in an Ave1‐dependent manner. Thus, we demonstrate that the functionality of tomato Ve1 in Verticillium wilt resistance through recognition of the Verticillium effector Ave1 is retained after transfer to tobacco and cotton, implying that the Ve1‐mediated immune signalling pathway is evolutionary conserved across these plant species. Moreover, our results suggest that transfer of tomato Ve1 across sexually incompatible plant species can be exploited in breeding programmes to engineer Verticillium wilt resistance.

Comparative analysis of the predicted secretomes of Rosaceae scab pathogens Venturia inaequalis and V. pirina reveals expanded effector families and putative determinants of host range

BackgroundFungal plant pathogens belonging to the genus Venturia cause damaging scab diseases of members of the Rosaceae. In terms of economic impact, the most important of these are V. inaequalis, which infects apple, and V. pirina, which is a pathogen of European pear. Given that Venturia fungi colonise the sub-cuticular space without penetrating plant cells, it is assumed that effectors that contribute to virulence and determination of host range will be secreted into this plant-pathogen interface. Thus the predicted secretomes of a range of isolates of Venturia with distinct host-ranges were interrogated to reveal putative proteins involved in virulence and pathogenicity.ResultsGenomes of Venturia pirina (one European pear scab isolate) and Venturia inaequalis (three apple scab, and one loquat scab, isolates) were sequenced and the predicted secretomes of each isolate identified. RNA-Seq was conducted on the apple-specific V. inaequalis isolate Vi1 (in vitro and infected apple leaves) to highlight virulence and pathogenicity components of the secretome. Genes encoding over 600 small secreted proteins (candidate effectors) were identified, most of which are novel to Venturia, with expansion of putative effector families a feature of the genus. Numerous genes with similarity to Leptosphaeria maculans AvrLm6 and the Verticillium spp. Ave1 were identified. Candidates for avirulence effectors with cognate resistance genes involved in race-cultivar specificity were identified, as were putative proteins involved in host-species determination. Candidate effectors were found, on average, to be in regions of relatively low gene-density and in closer proximity to repeats (e.g. transposable elements), compared with core eukaryotic genes.ConclusionsComparative secretomics has revealed candidate effectors from Venturia fungal plant pathogens that attack pome fruit. Effectors that are putative determinants of host range were identified; both those that may be involved in race-cultivar and host-species specificity. Since many of the effector candidates are in close proximity to repetitive sequences this may point to a possible mechanism for the effector gene family expansion observed and a route to diversification via transposition and repeat-induced point mutation.

Community of endophytic fungi from the medicinal and edible plant Fagopyrum tataricum and their antimicrobial activity

Purpose: To isolate and identify the fungal endophytes of Fagopyrum tataricum and assess their antimicrobial activity.Methods: The fungal isolates were identified according to their morphological characters and internal transcribed spacer (ITS) sequence analysis. Both broth-dilution-colorimetric assay and spore germination test were employed to assess the antimicrobial activity of fungal mycelia extracts.Results: A total of 72 endophytic fungal isolates were successfully obtained from the healthy tissues of F. tataricum. On the basis of their morphological and molecular characteristics, thirteen distinct fungal isolates were identified, and belonged to Alternaria, Bionectria, Botryosphaeria, Fusarium, Guignardia, Nectria, Neonectria, Phomopsis, Pseudocercospora and Verticillium spp. Of these, Pseudocercospora owned the maximum colonization frequency (15.3 %), next for Alternaria (12.5 %) and Neonectria (11.1 %). Through antimicrobial screening assays, six endophytes (Fataf-4, 6, 9, 11, 12 and 15) were active against Bacillus subtilis, Staphylococcus aureus, Agrobacterium tumefaciens, Escherichia coli and Pseudomonas lachrymans, and their minimal inhibitory concentration (MIC) ranged from 0.125 to 2.00 mg/mL. Furthermore, the endophytes, Fataf-9 and Fataf-11, also displayed strong inhibitory activity on the spore germination of F. oxysporum f. sp. vasinfectum and F. oxysporum f. sp. cucumerinum, and their median effective inhibitory concentration (IC50) was 0.356, 0.463, 0.451 and 0.489 mg/mL, respectively.Conclusion: The endophytic fungi of F. tataricum appear to be diverse and promising in their antimicrobial activity, and may represent a potential source of antibiotics for agriculture and/or pharmaceutical applications.Keywords: Endophytic fungi, Fagopyrum tataricum, Diversity, Antimicrobial activity, Antibiotics, Pseudocercospora

Effects of soil amendments combined with solarization on the soil microbial community in strawberry cultivation using quantitative real-time PCR

Experiments were conducted in commercial fields of strawberry-growing areas of the Aydin Province of Turkey during two cropping seasons: 2010–2011 and 2011–2012. Each year, eight separate treatments were used: (1) untreated control (C), (2) solarization alone (S), (3) solarization + broccoli (SBr), (4) solarization + fava bean (SFB), (5) solarization + dry olive-mill wastewater (SDOMW), (6) solarization + rice hulls (SRH), (7) solarization + sulfur powder (SSu), and (8) solarization + vermicast (SVe). DNA was extracted from bulk soil samples before and after solarization. The populations of the major taxonomic groups of bacteria and soil-borne fungal pathogens of strawberry were quantitatively calculated by quantitative real-time PCR (qPCR) with specific primer pairs using standard curves. The marketable fruit yield was recorded in the experimental plots. After the 6-week solarization period, there were significant reductions for total bacteria and for α-Proteobacteria in all of the experimental plots, except for SDOMW. However, the abundance of β-Proteobacteria significantly increased in all of the experimental plots (except for C and SFB in 2011). Significant increases in Firmicutes and Actinobacteria were also recorded in all of the treated plots. The highest significant increase was noticed with SDOMW treatments for total bacteria, α-Proteobacteria, β-Proteobacteria, Firmicutes and Actinobacteria in both years. The target level of Verticillium spp. Rhizoctonia solani and Fusarium oxysporum decreased significantly in all of the treated plots after solarization in both years. The reductions of M. phaseolina were significant in S, SVe, and SSu and were the highest in SDOMW in 2011; these reductions were significant in S, SSu and SDOMW in 2012. The abundance of Trichoderma spp. decreased insignificantly in all of the experimental plots. The highest significant yields were obtained from the combination of SDOMW, SRH and SSu compared to solarization alone in both seasons.

Identification of Novel Virulence-Associated Proteins Secreted to Xylem by Verticillium nonalfalfae During Colonization of Hop Plants.

Plant pathogens employ various secreted proteins to suppress host immunity for their successful host colonization. Identification and characterization of pathogen-secreted proteins can contribute to an understanding of the pathogenicity mechanism and help in disease control. We used proteomics to search for proteins secreted to xylem by the vascular pathogen Verticillium nonalfalfae during colonization of hop plants. Three highly abundant fungal proteins were identified: two enzymes, α-N-arabinofuranosidase (VnaAbf4.216) and peroxidase (VnaPRX1.1277), and one small secreted hypothetical protein (VnaSSP4.2). These are the first secreted proteins so far identified in xylem sap following infection with Verticillium spp. VnaPRX1.1277, classified as a heme-containing peroxidase from Class II, similar to other Verticillium spp. lignin-degrading peroxidases, and VnaSSP4.2, a 14-kDa cysteine-containing protein with unknown function and with a close homolog in related V. alfalfae strains, were further examined. The in planta expression of VnaPRX1.1277 and VnaSSP4.2 genes increased with the progression of colonization, implicating their role in fungal virulence. Indeed, V. nonalfalfae deletion mutants of both genes exhibited attenuated virulence on hop plants, which returned to the level of the wild-type pathogenicity in the knockout complementation lines, supporting VnaPRX1.1277 and VnaSSP4.2 as virulence factors required to promote V. nonalfalfae colonization of hop plants.

PCR detection of Ralstonia solanacearum: A new approach for rapid detection of bacterium from infected plants

Ralstonia solanacearum (Smith) is a soil-borne plant pathogen responsible for causing bacterial wilt and having wide host range which includes monocots, dicots, annual plants/trees and shrubs. It is a most destructive disease of solanceaous crops and ginger in north eastern region of India. The pathogen is primarily present in soils as saprophytic bacterium and it has ability to survive for long periods of time in various natural habitats. The bacterium causes sudden wilting in plants and difficult to detect at the initial level as similar symptom may also occur with many fungal organisms like Fusarium spp. and Verticillium spp. An attempt was made to develop a PCR-based rapid method for detection of this pathogen. This method requires only 3-5 hours against the conventional methods which generally require minimum 3 days to detect the pathogen. The PCR uses previously reported primer pairs for fliC gene (Rsol_fliC), which amplify 400bp region of fliC gene. The bacterial ooze from infected tissues was directly used as a source of DNA. The amplified product was cloned and sequenced for confirmation. The PCR based method developed in this report is very simple, robust and inexpensive and was successfully tested on four infected samples and further validated on over 50 samples of tomato which were infected by R. solanacearum.

Comparison of methods for detecting fungi in Silybum marianum (L.) Gaertn. seeds

ABSTRACT Milk thistle (Silybum marianum (L.) Gaertn.) is one of the most important medicinal plants. The fungi infesting its seeds may negatively influence their germination and health-promoting properties. However, there is no standard method for S. marianum seed health testing. The aim of this study was to find a suitable method for the detection and identification of fungi in/on milk thistle seeds. The following tests were used: deep-freeze blotter test, blotter test with an addition of mannitol, blotter test with an addition of polyethylene glycol, agar test on potato-dextrose agar (PDA), agar test on PDA after seed disinfection, agar test on reduced PDA and an agar test on reduced PDA after seed disinfection. Seeds were incubated for 10 and 14 days. The most prevalent fungi were: Alternaria alternata, Botrytis cinerea, Cladosporium spp., Fusarium spp., Penicillium spp., Rhizopus nigricans and Verticillium spp. A deep-freeze blotter test followed by a blotter test with an addition of mannitol and a blotter test with an addition of polyethylene glycol performed for 14 days could be recommended for the further study of milk thistle seed health testing, as they favoured the growth of the most important fungi.

PM 7/42 (2) Clavibacter michiganensis subsp. michiganensis

Specific scopeThis standard describes a diagnostic protocol for Clavibacter michiganensis subsp. michiganensis.Specific approval and amendmentApproved in 2004‐09.Revision adopted in 2012‐09.