Effects of Long-term Cotton Continuous Cropping on Soil Microbiome.

Verticillium wilt is a severe disease of cotton crops in Xinjiang and affecting yields and quality, due to the continuous cotton cropping in the past decades. The relationship between continuous cropping and the changes induced on soil microbiome remains unclear to date. In this study, the culture types of 15 isolates from Bole (5F), Kuitun (7F), and Shihezi (8F) of north Xinjiang were sclerotium type. Only isolates from field 5F belonged to nondefoliating pathotype, the others belonged to defoliating pathotype. The isolates showed pathogenicity differentiation in cotton. Fungal and bacterial communities in soils had some difference in alpha-diversity, relative abundance, structure and taxonomic composition, but microbial groups showed similarity in the same habitat, despite different sampling sites. The fungal phyla Ascomycota, and the bacterial phyla Proteobacteria, Actinobacteria, Chloroflexi, Acidobacteria and Gemmatimonadetes were strongly enriched. Verticillium abundance was significantly and positively correlated with AN, but negatively correlated with soil OM, AK and pH. Moreover, Verticillium was correlated in abundances with 5 fungal and 6 bacterial genera. Overall, we demonstrate that soil microbiome communities have similar responses to long-term continuous cotton cropping, providing new insights into the effects of continuous cotton cropping on soil microbial communities.

Rhizosphere fungal community structure succession of Xinjiang continuously cropped cotton.

The large-scale long-term plantation of cotton in the Xinjiang region has been accompanied by a regular and wide outbreak of soil-borne fungal diseases such as verticillium wilt, which significantly damaged the local cotton industry. High-throughput sequencing data showed that the cotton field cultivation management measures pose a significant influence upon the original ecological soil fungal community structure. During long-term continuous cropping of cotton, a new soil fungal community structure emerges after several repeated adjustments over five years. The number of verticillium wilt pathogens in the soil increased rapidly with prolonged continuous cropping time, reaching a maximum at around the 10th y; moreover, the abundance of the verticillium wilt pathogen only serves as one of numerous essential factors for disease occurrence. The fungal community structure and the abundance of verticillium wilt pathogens in local cotton fields are gradually formed under joint effects of year-long continuous cropping and supporting cultivation management measures.

From pathogen to endophyte: an endophytic population of Verticillium dahliae evolved from a sympatric pathogenic population.

The fungus Verticillium dahliae causes wilts of several hundred plant species, including potato and mint. Verticillium spp. also colonize sympatric hosts such as mustards and grasses as endophytes. The evolutionary history of and interactions between pathogenic and endophytic of this fungus are unknown. Verticillium dahliae isolates recovered from sympatric potato, mint, mustard and grasses were characterized genotypically with microsatellite markers and phenotypically for pathogenicity. The evolutionary history of pathogenic and endophytic populations was reconstructed and gene flow between populations quantified. Verticillium dahliae was recovered from all hosts. Endophytic populations were genetically and genotypically similar to but marginally differentiated from the potato population, from which they evolved. Bidirectional migration was detected between these populations and endophytic isolates were pathogenic to potato and behaved as endophytes in mustard and barley. Verticillium dahliae colonizes plants as both endophytes and pathogens. A historical host-range expansion together with endophytic and pathogenic capabilities are likely to have enabled infection of and gene flow between asymptomatic and symptomatic host populations despite minor differentiation. The ability of hosts to harbor asymptomatic infections and the stability of asymptomatic infections over time warrants investigation to elucidate the mechanisms involved in the maintenance of endophytism and pathogenesis.

Fungal Competitors Affect Production of Antimicrobial Lipopeptides in Bacillus subtilis Strain B9-5.

Bacillus subtilis has shown success in antagonizing plant pathogens where strains of the bacterium produce antimicrobial cyclic lipopeptides (CLPs) in response to microbial competitors in their ecological niche. To gain insight into the inhibitory role of these CLPs, B. subtilis strain B9-5 was co-cultured with three pathogenic fungi. Inhibition of mycelial growth and spore germination was assessed and CLPs produced by B. subtilis B9-5 were quantified over the entire period of microbial interaction. B. subtilis B9-5 significantly inhibited mycelial growth and spore germination of Fusarium sambucinum and Verticillium dahliae, but not Rhizopus stolonifer. LC-MS analysis revealed that B. subtilis differentially produced fengycin and surfactin homologs depending on the competitor. CLP quantification suggested that the presence of Verticillium dahliae, a fungus highly sensitive to the compounds, caused an increase followed by a decrease in CLP production by the bacterium. In co-cultures with Fusarium sambucinum, a moderately sensitive fungus, CLP production increased more gradually, possibly because of its slower rate of spore germination. With co-cultures of the tolerant fungus Rhizopus stolonifer, B. subtilis produced high amounts of CLPs (per bacterial cell) for the duration of the interaction. Variations in CLP production could be explained, in part, by the pathogens' overall sensitivities to the bacterial lipopeptides and/or the relative growth rates between the plant pathogen and B. subtilis. CLP production varied substantially temporally depending on the targeted fungus, which provides valuable insight concerning the effectiveness of B. subtilis B9-5 protecting its ecological niche against the ingress of these pathogens.

Differentiation of mixed soil-borne fungi in the genus level using infrared spectroscopy and multivariate analysis.

Early detection of soil-borne pathogens, which have a negative effect on almost all agricultural crops, is crucial for effective targeting with the most suitable antifungal agents and thus preventing and/or reducing their severity. They are responsible for severe diseases in various plants, leading in many cases to substantial economic losses. In this study, infrared (IR) spectroscopic method, which is known as sensitive, accurate and rapid, was used to discriminate between different fungi in a mixture was evaluated. Mixed and pure samples of Colletotrichum, Verticillium, Rhizoctonia, and Fusarium genera were measured using IR microscopy. Our spectral results showed that the best differentiation between pure and mixed fungi was obtained in the 675-1800 cm-1 wavenumber region. Principal components analysis (PCA), followed by linear discriminant analysis (LDA) as a linear classifier, was performed on the spectra of the measured classes. Our results showed that it is possible to differentiate between mixed-calculated categories of phytopathogens with high success rates (~100%) when the mixing percentage range is narrow (40-60) in the genus level; when the mixing percentage range is wide (10-90), the success rate exceeded 85%. Also, in the measured mixed categories of phytopathogens it is possible to differentiate between the different categories with ~100% success rate.

Analysis of the hybrid genomes of two field isolates of the soil-borne fungal species Verticillium longisporum.

BACKGROUND: Brassica plant species are attacked by a number of pathogens; among them, the ones with a soil-borne lifestyle have become increasingly important. Verticillium stem stripe caused by Verticillium longisporum is one example. This fungal species is thought to be of a hybrid origin, having a genome composed of combinations of lineages denominated A and D. In this study we report the draft genomes of 2 V. longisporum field isolates sequenced using the Illumina technology. Genomic characterization and lineage composition, followed by selected gene analysis to facilitate the comprehension of its genomic features and potential effector categories were performed. RESULTS: The draft genomes of 2 Verticillium longisporum single spore isolates (VL1 and VL2) have an estimated ungapped size of about 70 Mb. The total number of protein encoding genes identified in VL1 was 20,793, whereas 21,072 gene models were predicted in VL2. The predicted genome size, gene contents, including the gene families coding for carbohydrate active enzymes were almost double the numbers found in V. dahliae and V. albo-atrum. Single nucleotide polymorphisms (SNPs) were frequently distributed in the two genomes but the distribution of heterozygosity and depth was not independent. Further analysis of potential parental lineages suggests that the V. longisporum genome is composed of two parts, A1 and D1, where A1 is more ancient than the parental lineage genome D1, the latter being more closer related to V. dahliae. Presence of the mating-type genes MAT1-1-1 and MAT1-2-1 in the V. longisporum genomes were confirmed. However, the MAT genes in V. dahliae, V. albo-atrum and V. longisporum have experienced extensive nucleotide changes at least partly explaining the present asexual nature of these fungal species. CONCLUSIONS: The established draft genome of V. longisporum is comparatively large compared to other studied ascomycete fungi. Consequently, high numbers of genes were predicted in the two V. longisporum genomes, among them many secreted proteins and carbohydrate active enzyme (CAZy) encoding genes. The genome is composed of two parts, where one lineage is more ancient than the part being more closely related to V. dahliae. Dissimilar mating-type sequences were identified indicating possible ancient hybridization events.

Detection of Verticillium species in Swedish soils using real-time PCR.

Verticillium species are soilborne plant pathogens, responsible for big yield losses worldwide. Here, we report improved procedures to generate DNA from Verticillium species imbedded in farm soils. Using new genomic sequence information, primers for V. dahliae, V. albo-atrum, V. tricorpus, and V. longisporum were designed. In a survey of 429 samples from intensively farmed soil of two Swedish regions, only V. dahliae and V. longisporum were identified. A bias towards V. longisporum (40%) was seen in the south, whereas V. dahliae was more frequent in the western region (19%). Analyses of soil and leaf samples from 20 sugar beet fields, where foliar wilting had been observed, revealed V. dahliae DNA in all leaf and soil samples and V. longisporum in 18 soil samples, illustrating host choice and longevity of the V. longisporum microsclerotia. This study demonstrates the applicability of new molecular diagnostic tools that are important for growers of variable crops.

Clonal Expansion and Migration of a Highly Virulent, Defoliating Lineage of Verticillium dahliae.

We used a population genomics approach to test the hypothesis of clonal expansion of a highly fit genotype in populations of Verticillium dahliae. This fungal pathogen has a broad host range and can be dispersed in contaminated seed or other plant material. It has a highly clonal population structure, with several lineages having nearly worldwide distributions in agricultural crops. Isolates in lineage 1A are highly virulent and cause defoliation in cotton, okra, and olive (denoted 1A/D), whereas those in other lineages cause wilting but not defoliation (ND). We tested whether the highly virulent lineage 1A/D could have spread from the southwestern United States to the Mediterranean basin, as predicted from historical records. We found 187 single-nucleotide polymorphisms (SNPs), determined by genotyping by sequencing, among 91 isolates of lineage 1A/D and 5 isolates in the closely related lineage 1B/ND. Neighbor-joining and maximum-likelihood analyses on the 187 SNPs showed a clear divergence between 1A/D and 1B/ND haplotypes. Data for only 77 SNPs were obtained for all 96 isolates (no missing data); lineages 1A/D and 1B/ND differed by 27 of these 77 SNPs, confirming a clear divergence between the two lineages. No evidence of recombination was detected within or between these two lineages. Phylogenetic and genealogical analyses resulted in five distinct subclades of 1A/D isolates that correlated closely with geographic origins in the Mediterranean basin, consistent with the hypothesis that the D pathotype was introduced at least five times in independent founder events into this region from a relatively diverse source population. The inferred ancestral haplotype was found in two isolates sampled before 1983 from the southwestern United States, which is consistent with historical records that 1A/D originated in North America. The five subclades coalesce with the ancestral haplotype at the same time, consistent with a hypothesis of rapid population expansion in the source population during the emergence of 1A/D as a severe pathogen of cotton in the United States.

Globally invading populations of the fungal plant pathogen Verticillium dahliae are dominated by multiple divergent lineages.

The spread of aggressive fungal pathogens into previously non-endemic regions is a major threat to plant health and food security. Analyses of the spatial and genetic structure of plant pathogens offer valuable insights into their origin, dispersal mechanisms and evolution, and have been useful to develop successful disease management strategies. Here, we elucidated the genetic diversity, population structure and demographic history of worldwide invasion of the ascomycete Verticillium dahliae, a soil-borne pathogen, using a global collection of 1100 isolates from multiple plant hosts and countries. Seven well-differentiated genetic clusters were revealed through discriminant analysis of principal components (DAPC), but no strong associations between these clusters and host/geographic origin of isolates were found. Analyses of clonal evolutionary relationships among multilocus genotypes with the eBURST algorithm and analyses of genetic distances revealed that genetic clusters represented several ancient evolutionary lineages with broad geographic distribution and wide host range. Comparison of different scenarios of demographic history using approximate Bayesian computations revealed the branching order among the different genetic clusters and lineages. The different lineages may represent incipient species, and this raises questions with respect to their evolutionary origin and the factors allowing their maintenance in the same areas and same hosts without evidence of admixture between them. Based on the above findings and the biology of V. dahliae, we conclude that anthropogenic movement has played an important role in spreading V. dahliae lineages. Our findings have implications for the development of management strategies such as quarantine measures and crop resistance breeding.

Frequency of Verticillium Species in Commercial Spinach Fields and Transmission of V. dahliae from Spinach to Subsequent Lettuce Crops.

Verticillium wilt caused by V. dahliae is a devastating disease of lettuce in California (CA). The disease is currently restricted to a small geographic area in central coastal CA, even though cropping patterns in other coastal lettuce production regions in the state are similar. Infested spinach seed has been implicated in the introduction of V. dahliae into lettuce fields but direct evidence linking this inoculum to wilt epidemics in lettuce is lacking. In this study, 100 commercial spinach fields in four coastal CA counties were surveyed to evaluate the frequency of Verticillium species recovered from spinach seedlings and the area under spinach production in each county was assessed. Regardless of the county, V. isaacii was the most frequently isolated species from spinach followed by V. dahliae and, less frequently, V. klebahnii. The frequency of recovery of Verticillium species was unrelated to the occurrence of Verticillium wilt on lettuce in the four counties but was related to the area under spinach production in individual counties. The transmission of V. dahliae from infested spinach seeds to lettuce was investigated in microplots. Verticillium wilt developed on lettuce following two or three plantings of Verticillium-infested spinach, in independent experiments. The pathogen recovered from the infected lettuce from microplots was confirmed as V. dahliae by polymerase chain reaction assays. In a greenhouse study, transmission of a green fluorescence protein-tagged mutant strain of V. dahliae from spinach to lettuce roots was demonstrated, after two cycles of incorporation of infected spinach residue into the soil. This study presents conclusive evidence that V. dahliae introduced via spinach seed can cause Verticillium wilt in lettuce.

Diversity and biotransformative potential of endophytic fungi associated with the medicinal plant Kadsura angustifolia.

This study investigated the diversity and host component-transforming activity of endophytic fungi in medicinal plant Kadsura angustifolia. A total of 426 isolates obtained were grouped into 42 taxa belonging to Fungi Imperfecti (65.96%), Ascomycota (27.00%), Zygomycota (1.64%), Basidiomycota (0.47%) and Mycelia Sterilia (4.93%). The abundance, richness, and species composition of endophytic assemblages were significantly dependent on the tissue and the sampling site. Many phytopathogenic species associated with healthy K. angustifolia were found prevalent. Among them, Verticillium dahliae was dominant with 16.43% abundance. From 134 morphospecies selected, 39 showed remarkable biocatalytic activity and were further identified as species belonging to the genera Colletotrichum, Eupenicillium, Fusarium, Hypoxylon, Penicillium, Phomopsis, Trametes, Trichoderma, Umbelopsis, Verticillium and Xylaria on the basis of the sequence analysis of the internal transcribed spacer (ITS1-5.8S-ITS2). The results obtained in this work show that K. angustifolia is an interesting reservoir of pathogenic fungal species, and could be a community model for further ecological and evolutionary studies. Additionally, the converting potency screening of some endophytic fungi from this specific medicinal plant may provide an interesting niche on the search for novel biocatalysts.

Influence of soil type, cultivar and Verticillium dahliae on the structure of the root and rhizosphere soil fungal microbiome of strawberry.

Sustainable management of crop productivity and health necessitates improved understanding of the ways in which rhizosphere microbial populations interact with each other, with plant roots and their abiotic environment. In this study we examined the effects of different soils and cultivars, and the presence of a soil-borne fungal pathogen, Verticillium dahliae, on the fungal microbiome of the rhizosphere soil and roots of strawberry plants, using high-throughput pyrosequencing. Fungal communities of the roots of two cultivars, Honeoye and Florence, were statistically distinct from those in the rhizosphere soil of the same plants, with little overlap. Roots of plants growing in two contrasting field soils had high relative abundance of Leptodontidium sp. C2 BESC 319 g whereas rhizosphere soil was characterised by high relative abundance of Trichosporon dulcitum or Cryptococcus terreus, depending upon the soil type. Differences between different cultivars were not as clear. Inoculation with the pathogen V. dahliae had a significant influence on community structure, generally decreasing the number of rhizosphere soil- and root-inhabiting fungi. Leptodontidium sp. C2 BESC 319 g was the dominant fungus responding positively to inoculation with V. dahliae. The results suggest that 1) plant roots select microorganisms from the wider rhizosphere pool, 2) that both rhizosphere soil and root inhabiting fungal communities are influenced by V. dahliae and 3) that soil type has a stronger influence on both of these communities than cultivar.

Multiplex real-time PCR assays for detection of four seedborne spinach pathogens.

AIMS: To develop multiplex TaqMan real-time PCR assays for detection of spinach seedborne pathogens that cause economically important diseases on spinach. METHODS AND RESULTS: Primers and probes were designed from conserved sequences of the internal transcribed spacer (for Peronospora farinosa f. sp. spinaciae and Stemphylium botryosum), the intergenic spacer (for Verticillium dahliae) and the elongation factor 1 alpha (for Cladosporium variabile) regions of DNA. The TaqMan assays were tested on DNA extracted from numerous isolates of the four target pathogens, as well as a wide range of nontarget, related fungi or oomycetes and numerous saprophytes commonly found on spinach seed. Multiplex real-time PCR assays were evaluated by detecting two or three target pathogens simultaneously. Singular and multiplex real-time PCR assays were also applied to DNA extracted from bulked seed and single spinach seed. CONCLUSIONS: The real-time PCR assays were species-specific and sensitive. Singular or multiplex real-time PCR assays could detect target pathogens from both bulked seed samples as well as single spinach seed. SIGNIFICANCE AND IMPACT OF THE STUDY: The freeze-blotter assay that is currently routinely used in the spinach seed industry to detect and quantify three fungal seedborne pathogens of spinach (C. variabile, S. botryosum and V. dahliae) is quite laborious and takes several weeks to process. The real-time PCR assays developed in this study are more sensitive and can be completed in a single day. As the assays can be applied easily for routine seed inspections, these tools could be very useful to the spinach seed industry.

Molecular mapping and validation of a major QTL conferring resistance to a defoliating isolate of verticillium wilt in cotton (Gossypium hirsutum L.).

Verticillium wilt (VW) caused by Verticillium dahliae Kleb is one of the most destructive diseases of cotton. Development and use of a VW resistant variety is the most practical and effective way to manage this disease. Identification of highly resistant genes/QTL and the underlining genetic architecture is a prerequisite for developing a VW resistant variety. A major QTL qVW-c6-1 conferring resistance to the defoliating isolate V991 was identified on chromosome 6 in LHB22×JM11 F2∶3 population inoculated and grown in a greenhouse. This QTL was further validated in the LHB22×NNG F2∶3 population that was evaluated in an artificial disease nursery of V991 for two years and in its subsequent F4 population grown in a field severely infested by V991. The allele conferring resistance within the QTL qVW-c6-1 region originated from parent LHB22 and could explain 23.1-27.1% of phenotypic variation. Another resistance QTL qVW-c21-1 originated from the susceptible parent JM11 was mapped on chromosome 21, explaining 14.44% of phenotypic variation. The resistance QTL reported herein provides a useful tool for breeding a cotton variety with enhanced resistance to VW.

Verticillium dahliae race 2-specific PCR reveals a high frequency of race 2 strains in commercial spinach seed lots and delineates race structure.

Two pathogenic races of Verticillium dahliae have been described on lettuce and tomato. Host resistance to race 1 is governed by plant immune receptors that recognize the race 1-specific fungal effector Ave1. Only partial resistance to race 2 exists in lettuce. Although polymerase chain reaction (PCR) assays are available to identify race 1, no complementary test exists to positively identify race 2, except for lengthy pathogenicity assays on host differentials. Using the genome sequences of two isolates of V. dahliae, one each from races 1 and 2, we identified potential markers and PCR primers to distinguish the two races. Several primer pairs based on polymorphisms between the races were designed and tested on reference isolates of known race. One primer pair, VdR2F-VdR2R, consistently yielded a 256-bp amplicon in all race 2 isolates exclusively. We screened DNA from 677 V. dahliae isolates, including 340 from spinach seedlots, with the above primer pair and a previously published race 1-specific primer pair. DNA from isolates that did not amplify with race 1-specific PCRs amplified with the race 2-specific primers. To validate this, two differential lines of lettuce were inoculated with 53 arbitrarily selected isolates from spinach seed and their pathogenicity and virulence were assessed in a greenhouse. The reactions of the differential cultivars strongly supported the PCR data. V. dahliae race structure was investigated in crops in coastal California and elsewhere using primers specific to the two races. All artichoke isolates from California were race 1, whereas nearly all tomato isolates were race 2. Isolates from lettuce, pepper, and strawberry from California as well as isolates from spinach seed from two of four countries comprised both races, whereas only race 2 was observed in cotton, mint, olive, and potato. This highlights the importance of identifying resistance against race 2 in different hosts. The technique developed in this study will benefit studies in ecology, population biology, disease surveillance, and epidemiology at local and global scales, and resistance breeding against race 2 in lettuce and other crops.

Clonal expansion of Verticillium dahliae in lettuce.

Few studies in population biology have documented how structure and diversity of pathogens evolve over time at local scales. With the historical samples of Verticillium dahliae available from lettuce, we investigated the structure and diversity of this pathogen in time and space. Three hundred twenty-nine V. dahliae isolates from lettuce fields collected over 18 years were characterized with polymorphic microsatellite markers and polymerase chain reaction tests for race and mating type. Genetic variation within and among commercial lettuce fields in a single season was also investigated using an additional 146 isolates. Sixty-two haplotypes (HTs) were observed among the 329 isolates. A single HT was frequently observed over multiple years and locations (61.40%). Genetic diversity, allelic richness, and private allelic richness suggested a relatively recent clonal expansion. Race 1 (93.63%) and MAT1-2-1 (99.69%) were overwhelmingly represented among the isolates. Linkage disequilibrium was significant (P < 0.001) for all populations, suggesting limited sexual recombination in the sampled populations from lettuce. Populations from 2006, 2009, and 2010 had higher numbers of unique HTs, implying a recent introduction of novel HTs. We conclude that V. dahliae population from lettuce evaluated in this study is expanding clonally, consistent with an asexually reproducing pathogen, and the movement of clonal genotypes locally occurs over time.

A novel and rapid loop-mediated isothermal amplification assay for the specific detection of Verticillium dahliae.

AIMS: In this study, a loop-mediated isothermal amplification (LAMP) assay has been developed and evaluated for the rapid and sensitive detection of Verticillium dahliae Kleb., the causal agent of vascular wilts in many economically important crops. METHODS AND RESULTS: LAMP primers were designed based on a previously described RAPD marker, and the LAMP assay was applied for direct detection of V. dahliae grown on medium and from soil samples without DNA purification steps (direct-LAMP). Thirty-two agricultural soil samples from various olive orchards were collected, and the presence of pathogen was detected by LAMP, direct-LAMP and nested-PCR methods. The LAMP methodology could successfully detect V. dahliae with high specificity, and cross-reaction was not observed with different pathogenic and nonpathogenic fungi and bacteria. The LAMP assay was capable of detecting a minimum of 500 and 50 fg of purified target DNA per reaction of V. dahliae ND and D pathotypes, respectively. In contrast, nested-PCR could only detect 5 pg reaction(-1) for both pathotypes. In artificially infested soil samples, the LAMP method detected 5 microsclerotia per gram of soil. Conversely, nested-PCR assay detected 50 microsclerotia g(-1) soil. The detection ratios of LAMP and direct-LAMP protocols were better (26 and 24 positive samples out of 32 agricultural soils analysed, respectively) than that obtained for nested-PCR method (22 positive results). Moreover, direct-LAMP yielded positive detection of V. dahliae in agricultural soil samples within 60-80 min. CONCLUSIONS: The newly developed LAMP method was proved to be an effective, simple and rapid method to detect V. dahliae without the need for either expensive equipment or DNA purification. SIGNIFICANCE AND IMPACT OF STUDY: This technique can be considered as an excellent standard alternative to plating and nested-PCR assays for the early, sensitive and low-cost detection of V. dahliae as well as other soilborne pathogens in the field.

Comparative pathogenicity, biocontrol efficacy, and multilocus sequence typing of Verticillium nonalfalfae from the invasive Ailanthus altissima and other hosts.

Verticillium wilt, caused by Verticillium nonalfalfae, is currently killing tens of thousands of highly invasive Ailanthus altissima trees within the forests in Pennsylvania, Ohio, and Virginia and is being considered as a biological control agent of Ailanthus. However, little is known about the pathogenicity and virulence of V. nonalfalfae isolates from other hosts on Ailanthus, or the genetic diversity among V. nonalfalfae from confirmed Ailanthus wilt epicenters and from locations and hosts not associated with Ailanthus wilt. Here, we compared the pathogenicity and virulence of several V. nonalfalfae and V. alfalfae isolates, evaluated the efficacy of the virulent V. nonalfalfae isolate VnAa140 as a biocontrol agent of Ailanthus in Pennsylvania, and performed multilocus sequence typing of V. nonalfalfae and V. alfalfae. Inoculations of seven V. nonalfalfae and V. alfalfae isolates from six plant hosts on healthy Ailanthus seedlings revealed that V. nonalfalfae isolates from hosts other than Ailanthus were not pathogenic on Ailanthus. In the field, 100 canopy Ailanthus trees were inoculated across 12 stands with VnAa140 from 2006 to 2009. By 2011, natural spread of the fungus had resulted in the mortality of >14,000 additional canopy Ailanthus trees, 10,000 to 15,000 Ailanthus sprouts, and nearly complete eradication of Ailanthus from several smaller inoculated stands, with the exception of a few scattered vegetative sprouts that persisted in the understory for several years before succumbing. All V. nonalfalfae isolates associated with the lethal wilt of Ailanthus, along with 18 additional isolates from 10 hosts, shared the same multilocus sequence type (MLST), MLST 1, whereas three V. nonalfalfae isolates from kiwifruit shared a second sequence type, MLST 2. All V. alfalfae isolates included in the study shared the same MLST and included the first example of V. alfalfae infecting a non-lucerne host. Our results indicate that V. nonalfalfae is host adapted and highly efficacious against Ailanthus and, thus, is a strong candidate for use as a biocontrol agent.

Production of cold-adapted cellulase by Verticillium sp. isolated from Antarctic soils

Background: Cellulose can be converted to ethanol by simultaneous saccharification and fermentation (SSF). The difference between the optimal temperature of cellulase and microbial fermentation, however, has been identified as the critical problem with SSF. In this study, one fungal strain (AnsX1) with high cellulase activity at low temperature was isolated from Antarctic soils and identified as Verticillium sp. by morphological and molecular analyses. Results: The biochemical properties of crude AnsX1 cellulase samples were studied by filter paper cellulase assay. The maximum cellulase activity was achieved at low temperature in an acidic environment with addition of metal ions. Furthermore, AnsX1 cellulase demonstrated 54-63% enzymatic activity at ethanol concentrations of 5-10%. AnsX1 cellulase production was influenced by inoculum size, carbon and nitrogen sources, and elicitors. The optimal culture conditions for AnsX1 cellulase production were 5% inoculum, wheat bran as carbon source, (NH4)2SO4 as nitrogen source, and sorbitol added in the medium. Conclusions: Our present work has potential to enable the development of an economic and efficient cold-adapted cellulase system for bioconversion of lignocellulosic biomass into biofuels in future.

Upward movement of Verticillium dahliae from soil to olive plants detected by qPCR.

Olive trees play an important role in cultural, ecological, environmental and social fields, constituting in large part the Mediterranean landscape. In Tuscany, an important economic activity is based on olive. Unfortunately, the Verticillium wilt affects this species and causes vascular disease. In the present study, a real-time quantitative PCR approach has been used to detect and quantify Verticillium dahliae in soil and in olive tree tissues both in micropropagated and in seedling olives. The minimum amounts of V. dahliae DNA sequences detected in soil were 11.4 fg which is equivalent to less than one fungal haploid genome. In micropropagated olive the pathogen was detected in the leaves after 43 days, showing a vertical upward movement of the fungus from the culture medium to stem and leaves. A similar fungal behaviour was observed in inoculated olive stem where after 15 days the fungal DNA was detected from symptomless stem tissue above 8 cm the inoculation site. The described molecular approach is expected to provide a more sensitive and less time-consuming alternative detection method for V. dahliae than plating assay procedures, which were traditionally proposed as an early diagnosis method for Verticillium wilt to farmers and tree nursery growers.