Genetic relationships between virulence,vegetative compatibility and ISSR marker of Verticillium dahliae isolated from cotton

Verticillium dahliae is one of the most important pathogens causing Verticillium wilt. We studied the characterisation of the genetic relationship between virulence, vegetative compatibility groups (VCGs) and inter-simple sequence repeat (ISSR). A total of 48 V. dahliae isolates, in which 16 are collected from different cotton growing regions in China and 4 isolates belonged to all known VCGs, are used. Half of them were found highly virulent. Mutants (565) were obtained using the nitrate non-utilising mutant. These mutants were grouped into three VCGs: VCG1 (27 isolates), VCG 2 (14 isolates) and VCG 3 (7 isolates). Use of ISSR indicated two main clusters that were related to VCG and virulence. Genetic diversity lineages were obviously correlated to VCGs and ISSRs according to their geographical origin, virulence and ISSR genetic variation. This study could be useful to design and develop effective management strategies beside for quarantine purposes on Verticillium wilt control.     

Host Specialization among Vegetative Compatibility Groups of Verticillium dahliae in Relation to Verticillium longisporum

A collection of 24 isolates of Verticillium dahliae and 10 isolates of Verticillium longisporum originating from nine different host plants and from several geographic regions was tested for host specificity on 11 economically important crops such as potato, tomato, strawberry, linseed, three legumes and four Brassica species. In order to reveal host specificity the potential of each isolate to induce disease and affect plant yield was recorded for all isolate–host combinations. The collected data were statistically processed by means of a cluster analysis. As a result, the host range of individual isolates was found to be more dependent on the vegetative compatibility group (VCG) of the isolate than on its original host plant provenance. Twenty‐two out of 24 V. dahliae isolates belonged to either VCG 2B or 4B. VCG 2B isolates showed specificity for legumes, strawberry, potato and linseed, whereas VCG 4B was specifically virulent on potato, strawberry and linseed. Subgroups within VCG 2B and 4B almost lacking any host preference were designated 2B* and 4B*. Three isolates from VCG 2B*, however, severely attacked tomato which is a host outside the authentic host range of VCG 2B. The pathogenicity of V. longisporum isolates was restricted to cruciferous hosts. Conversely, cruciferous plants were not affected by isolates from VCGs 2B and 4B of V. dahliae. This lack of cross‐infectivity of certain subpopulations of V. dahliae and of V. longisporum may be useful in the management of this soil‐borne wilt disease.     

High‐Throughput Assessment and Genetic Investigation of Vegetative Compatibility in Verticillium dahliae

Classification of isolates into vegetative compatibility groups (VCGs) using nitrate‐non‐utilizing (nit) mutants has been widely used for the characterization of Verticillium dahliae populations. However, certain methodological limitations prevent its application on a large scale. Furthermore, systematic investigations into the genetics underlying complementation tests between nit mutants of fungal isolates (i.e. heterokaryon formation) are lacking for Verticillium species. In this work, a diverse collection of 27 V. dahliae isolates – including representatives of all VCGs, both mating types, and heterokaryon self‐incompatible isolates – was employed for the development and optimization of (i) a protocol for the rapid generation of nit mutants of V. dahliae isolates using UV‐irradiation and (ii) a reproducible high‐throughput procedure for complementation tests between nit mutants in liquid cultures using 96‐well microplates. The genetic analysis of selected heterokaryons demonstrated that the frequently encountered ‘weak’ cross‐reactions between VCGs and their subgroups can be actually heterokaryotic, implying the absence of strict genetic barriers between VCGs. In conclusion, we provide in this work an optimized method for the high‐throughput VCG assignment of V. dahliae populations and a genetic analysis of heterokaryons that may have serious implications for the interpretation of VCG classification data. These advancements in the available methodology and the genetic background of vegetative compatibility grouping may contribute to a better understanding of the population biology of V. dahliae and possibly other mitosporic fungi.     

Detection of the defoliating and nondefoliating pathotypes of <Emphasis Type="Italic">Verticillium dahliae</Emphasis> in artificial and natural soils by nested PCR

In Spain, Verticillium wilt, caused by Verticillium dahliae, is the most important disease of cotton and olive. Isolates of V. dahliae infecting these crops can be classified into highly virulent, defoliating (D), and mildly virulent, nondefoliating (ND), pathotypes. Infested soil is the primary source of inoculum for Verticillium wilt epidemics in cotton and olive, and severity of disease relates to the prevailing V.dahliae pathotype. In this work we have adapted the use of previously developed primer pairs specific for D and ND V. dahliae for the detection of these pathotypes by nested PCR in artificial and natural soils. Success in the detection procedure depends upon efficiency in extracting PCR-quality DNA from soil samples. We developed an efficient DNA extraction method from microsclerotia infesting the soil that includes the use of acid washed sand during the grinding process and skimmed milk to avoid co-purification of Taq-polymerase inhibitors with DNA. The specific nested-PCR procedure effectively detected 10 or more microsclerotia per gram of soil. The detection procedure has proven efficient when used with a naturally infested soil, thus demonstrating usefullness of the diagnostic method for rapid and accurate assessment of soil contamination by V. dahliae pathotypes.     

How do Japanese isolates ofVerticillium dahliae correspond with standardized VCG testers?

We examined the vegetative compatibility of 56 Japanese isolates provisionally assigned to four subgroups ofV. dahliae to estimate the genetic relatedness with testers of the standardized VCGs. Subgroup J1 was assigned to VCG 2A/B as a new category of assignment. Subgroup J2, except isolate Vdt 110, was assigned to VCG 2A, and subgroup J3, except isolate Vdf 1, was assigned to VCG 2B. Isolates Vdf 1 and Vdt 110 were assigned to VCG 2A/B. Subgroup J4 was assigned to two subgroups, VCG 4B for Vde 1 and VCG 4A/B for FY 3 and HR 1. Four isolates were compatible with both VCG 2 and 4. Isolate U56 was compatible with VCG 2A/B and 4A. Isolates of VCG 2A, Vdt 9 and FF1, were compatible with either VCG 4A or 4A/B. One isolate of VCG 2B, Vdp-4, was compatible with VCG 4A. Three isolates of subgroup J2 showed weak reactions with the testers of VCG 4. These isolates may be “bridging strains”. Japanese isolates were composed of two VCGs, 2 and 4, “bridging strains” compatible with these VCGs, and some self-incopatible isolates. Testers of VCG 1 and VCG 3 did not show any reactions with the Japanese isolates.     

Application of RAPD-PCR for Virulence Type Analysis within Verticillium dahliae and V. longisporum

A collection of 24 isolates of Verticillium dahliae, 11 isolates of V. longisporum and one isolate of V. albo‐atrum originating from different host plants and geographical regions was tested for genetic variability by random amplified polymorphic DNA‐polymerase chain reaction (RAPD–PCR). Based on nine primers, the three Verticillium species could be clearly differentiated. Likewise, this analysis provided a distinct separation of vegetative compatibility groups (VCG) 2B, 4A and 4B of V. dahliae by specific DNA banding patterns. Additionally, V. longisporum was found to segregate into two subgroups with only 88% similarity. This molecular‐genetic approach was used for the analysis of randomly selected Verticillium isolates from a field with high intensity of oilseed rape cultivation (33% in crop rotation). RAPD‐PCR analysis revealed that 95 of 100 isolates tested belonged to V. longisporum and 5 to VCG 2B of V. dahliae. This study demonstrates an adaptation of Verticillium soil populations to a specific cropping history.     

Assessment of vegetative compatibility of race-2 tomato wilt isolates ofVerticillium dahliae in Japan

Verticillium dahliae race-2 can invade the resistant cultivars of tomato possessing theVe gene. This new race was recently found in several regions in Japan, and 10 isolates ofV. dahliae race-2 from these regions were used in our study. Pathogenicity tests identified these isolates as the tomato pathotype (B). We examined the vegetative compatibility of 8 of these 10 Japanese isolates ofV. dahliae race-2 to estimate their genetic relatedness with the testers of Japanese vegetative compatibility group previously proposed (VCGJ) usingnit mutants. Compatiblenit1 and NitM mutants were obtained from allV. dahliae race-2 isolates. Selected representativenit1 and NitM mutants of eachV. dahliae race-2 isolates were paired with VCGJ testers. All isolates ofV. dahliae race-2 showed a strong reaction with VCGJ2, i.e., tomato pathotype. All isolates ofV. dahliae race-2 except for isolate To22 reacted weakly to VCGJ1 and J3. Japanese isolates ofV. dahliae race-2 were assigned as VCGJ2 and were hence vegetatively closely related with those ofV. dahliae race-1. The origin of Japanese isolates ofV. dahliae race-2 was discussed.     

Antifungal activity of essential oils against three vegetative-compatibility groups of <Emphasis Type="Italic">Verticillium dahliae</Emphasis>

The antifungal activities of volatile phase effects of essential oils from Origanum onites, O. syriacum, O. minutiflorum, O. vulgare, O, marjorana, Thymus vulgaris, T. serpyllum, Rosmarinus officinalis, Salvia officinalis and Micromeria fruticosa were evaluated for their ability to inhibit growth of three vegetative compatibility groups (VCGs) of Verticillium dahliae. Carvacrol was the main component of O. onites, O. minutiflorum and O. vulgare essential oils, while γ-terpinene was the main component of O. syriacum. P-cymene and thymol were the dominant component of T. vulgaris and T. serpyllum. β- thujone and l-camphor were the main component of S. officinalis. Polegone and isomenthone were the dominant components of M. fruticosa essential oil. Based on the in vitro test, the degree of fungistatical effects can be ranked in the following order of inhibition: O. syriacum = O. onites = O. minutiflorum = O. vulgare = T. vulgaris > T. serpyllum > M. fruticosa > S. officinalis = O. marjorana > R. officinalis. The essential oils of S. officinalis, O. marjorana and R. officinalis displayed moderate antifungal activity, that increased with increasing concentrations. Among the VCGs, VCG2A and VCG4B were found to be highly sensitive to the essential oils. The essential oils of O. syriacum, O. onites, O. minutiflorum, O. vulgare and T. vulgaris were the most efficacious, demonstrating strong antifungal activity against all of the tested VCGs of V. dahliae at relatively low concentrations and they could find practical application as natural fungicides in the prevention and protection of plants from V. dahliae infections.     

Vegetative compatibility of an isolate ofVerticillium dahliae pathogenic to both tomato and pepper

An isolate ofVerticillum dahliae Vdp-4, pathogenic to both tomato and pepper (tomato-pepper pathotype), was examined for its vegetative compatibility with testers of the Japanese vegetative compatibility group (subgroups J1, J2, and J3). Seven isolates ofV. dahliae from the same field as Vdp-4 in Misato, Nagano Pref. and two isolates from Hokkaido were separately determined as either tomato pathotype (B) or pepper pathotype (C). Isolate 5922 previously reported as tomato-pepper pathotype was also examined. Compatiblenit1 and NitM mutants were obtained from all isolates except for isolates Vdp-3 and Vdt-10. The isolate of tomato-pepper pathotype Vdp-4 showed a strong reaction with VCGJ1 and J3 and was thus assigned to J3. Seven of these isolates showed compatibility and were assigned into three provisional subgroups. The isolate 5922 was self-incompatible.     

Yellow Vein Mosaic Disease in Okra (<i>Abelmoschus esculentus</i> L.): An Overview on Causal Agent,Vector and Management

Okra (Abelmoschus esculentus L.) belongs to the Malvaceae family and is one of the most essential and popular vegetables globally. It is rich in proteins, carbohydrates, and vitamins. Abiotic and biotic factors threaten okra productivity. Okra yellow vein mosaic disease (OYVMD) is the most destructive disease of okra. The causal agent, [(i.e., Okra yellow vein mosaic virus (OYVMV)] of this disease belongs to the family Geminiviridae and genus Begomovirus. OYVMV is a monopartite with additional ssDNA molecule. This virus has two components DNA-A for protein coding and DNA-B for symptoms induction. Whitefly transmits OYVMV in persistent manner. Characteristic symptoms of OYVMV infected okra plants are chlorosis, dwarfing, and yellowing of veins and fruits. High temperatures with moderate rainfall enhance the development of OYVMV disease and the whitefly population. However, high humidity with low temperature and rainfall has no significant role in developing the OYVMD and whitefly population. Moreover, the virus also affects the secondary metabolites in the infected okra plants. The virus can be managed through various strategies including the application of plant defense activators, the development of resistant varieties and by controlling its vector via pesticides and plant extracts. Various plant defense activators such as monopotassium phosphate (KH₂PO), salicylic acid, benzoic acid, and citric acid enhance resistance in okra against OYMVD. In addition, the resistance to OYMVD can also be achieved by successfully incorporating high yielding but resistant cultivars of acceptable quality. In this review, we have discussed history, economic impact, symptomology, disease development under a natural environment, genetics and management of OYVMV.     

Characterization of aflatoxigenic and non-aflatoxigenic <Emphasis Type="Italic">Aspergillus flavus</Emphasis> isolates from pistachio

Pistachio is a popular snack food. Aflatoxin contamination of pistachio nuts is a serious problem for many producing countries. The development of biological control methods based on ecological parameters is an environmentally friendly approach. Thirty-eight Aspergillus flavus isolates collected from a pistachio orchard in California (CA) were analyzed for production of aflatoxin (AF), cyclopiazonic acid (CPA), vegetative compatibility groups (VCGs), and mating types. All aflatoxigenic isolates produced both AFB₁ and CPA. The most toxigenic one was CA28 which produced 164 μg AFB₁ per 5 ml PDA fungal culture and small sclerotia (S strain, sclertoium size less than 400 μm). The other aflatoxigenic strains produce AFB₁ ranging from 1.2 μg to 80 μg per 5 ml fungal culture. Twenty-one percent of the CA isolates produced AFB₁, 84% produced CPA and half formed sclerotia on at least one of three tested media. The 38 CA isolates formed 26 VCGs, 6 of which had two or more isolates and 20 contained single isolates. The S strain isolates belong to 4 different VCGs. Genomic profiling by a retrotransposon DNA probe revealed fingerprint patterns that were highly polymorphic. The predicted VCGs (Pred-VCGs) based on a similarity coefficient >80% matched the VCGs of multiple isolates determined by complementation. All isolates within a VCG had the same mating-type gene of either MAT1-1 or MAT1-2. Uncorrected and VCG-corrected MAT1-1 and MAT1-2 among the isolates were equally distributed.     

Genome-Wide Identification of Candidate Genes Associated with <i>β</i>-glucan Traits in a Hulled and Hulless Barley (<i>Hordeum vulgare</i> L.) Population

Barley grain is a valuable source of β-glucan, which is an important component of dietary fiber with significant human health benefits. Although the genetic basis of β-glucan biosynthesis has been widely studied, a genome-wide association study (GWAS) is still required for a scan of the candidate genes related to the complex quantitative trait based on the high-quality barley reference genome. In this study, a GWAS was conducted using a population composed of 87 barley landraces (39 hulled and 48 hulless, β-glucan from 2.07% to 6.56%) with 191,098 nucleotide polymorphisms (SNPs) markers to cover the chromosomes with the highest density. The population was divided into four sub-populations (POP1~POP4), and the β-glucan content in POP2 was significantly higher than that in other groups, in which most of the hulless barley landraces are from Qinghai-Tibet Plateau in China. Among seven SNP markers identified by GWAS, two (SNP2 and SNP3) of them showed positive correlation to β-glucan trait and the remaining five (SNP1, SNP4, SNP5, SNP6 and SNP7) showed the negative relationship. Two candidate genes linked to SNP7, HORVU7Hr1G000320 and HORVU7Hr1G000040, belong to the nucleotide triphosphate hydrolase superfamily which is probable to affect the activities of β-glucan synthase. Another candidate gene associated with SNP1, HORVU1Hr1G000010, is possibly involved in sugar response. In conclusion, our results provide new insights into the genetic basis of β-glucan accumulation in barley grains, and the discovery of new SNP markers distributed in each chromosome and the associated candidate genes will be valuable for the breeding of functional barley varieties with high β-glucan.     

Verticillium wilt of pea (Pisum sativum)

A wilt disease of garden pea (Pisum sativum) caused by Verticillium dahliae is described and the range of pathogenicity of the isolate investigated. It is pathogenic to potato, sweet pea, antirrhinum and broad bean and isolates of V. dahliae from potato, lucerne and sweet pea and V. albo-atrum from lucerne are pathogenic to pea. Since the most common disease symptoms, acropetal progression of chlorosis and necrosis of the leaves followed by premature defoliation are indistinguishable from natural senescence, it is probable that disease and senescence symptoms are confused in the field. The premature defoliation results in marked reduction in green leaf area, leaf dry weight and pod yield.     

Identification of Pathogenicity-Related Genes in the Vascular Wilt Fungus <Emphasis Type="Italic">Verticillium dahliae</Emphasis> by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-Mediated T-DNA Insertional Mutagenesis

Verticillium dahliae is the causal agent of vascular wilt in many economically important crops worldwide. Identification of genes that control pathogenicity or virulence may suggest targets for alternative control methods for this fungus. In this study, Agrobacterium tumefaciens-mediated transformation (ATMT) was applied for insertional mutagenesis of V. dahliae conidia. Southern blot analysis indicated that T-DNAs were inserted randomly into the V. dahliae genome and that 69% of the transformants were the result of single copy T-DNA insertion. DNA sequences flanking T-DNA insertion were isolated through inverse PCR (iPCR), and these sequences were aligned to the genome sequence to identify the genomic position of insertion. V. dahliae mutants of particular interest selected based on culture phenotypes included those that had lost the ability to form microsclerotia and subsequently used for virulence assay. Based on the virulence assay of 181 transformants, we identified several mutant strains of V. dahliae that did not cause symptoms on lettuce plants. Among these mutants, T-DNA was inserted in genes encoding an endoglucanase 1 (VdEg-1), a hydroxyl-methyl glutaryl-CoA synthase (VdHMGS), a major facilitator superfamily 1 (VdMFS1), and a glycosylphosphatidylinositol (GPI) mannosyltransferase 3 (VdGPIM3). These results suggest that ATMT can effectively be used to identify genes associated with pathogenicity and other functions in V. dahliae.     

Morphological and Phylogenetic Resolution of <i>Diplodia neojuniperi</i> Emerging <i>Diplodia</i> Top Dieback of <i>Pinus thunbergii</i> Parl. in China

In Bazhong City, Sichuan Province, China, top dieback symptoms were found on many pine trees (Pinus thunbergii Parl). The tips of old needles first turned grayish-green and then developed into brown bands in the field. Phylogenetic analysis of concatenated ITS and EF1-α indicated the pathogen of this dieback disease as Diplodia neojuniperi. Additionally, effects of temperature, pH and medium on the mycelial growth were also characterized. The most favorable temperature and pH level for mycelial growth are 25°C and 8, respectively. The optimal medium for mycelial growth is PDA medium. To our knowledge, this is the first report of D. neojuniperi causes Diplodia top dieback on Pinus thunbergii. Our results provide fundamental information for monitoring and preventing such disease in the future.     

Vegetative compatibility relationships among weakly pathogenic isolates (pathotype E) ofVerticillium dahliae

Twenty-two isolates ofVerticillium dahliae, which were isolated from green soybeean (Glycine max), udo (Aralia cordata), horseradish (Cochlearia armoracia), sweetpea (Lathyrus odoratus), or a weed (Chenopodium album) were used in this study. Conidia and microsclerotia of these isolates were morphologically identical with those ofV. dahliae but did not coincide withV. longisporum. Pathogenicity tests showed that these isolates were of weak pathotype. Eleven of the 22 isolates, which were obtained from green soybean and udo, were pathogenic to green soybeans. Thus pathotype E was composed of two groups: ‘soybean pathotype’ which was pathogenic to green soybeans; and isolates nonpathogenic to green soybeans. The latter were defined as isolates of pathotype E in the narrow sense. Selected representativenit1 and NitM mutants of eachV. dahliae isolate were paired with VCGJ testers. Fourteen isolates ofV. dahliae (So1, So22, So23, So27, So28, So39, So40, So41, U54, U68, U69, U90, U95, and U115) showed complementary reactions with subgroups J1 and J3 and were assigned to subgroup J3. Isolate U108 was assigned to subgroup J2. Isolate HR1 was not compatible with any testers of VCGJ. With this exception, isolates of pathotype E in the narrow sense and those of ‘soybean pathotype’ were thus assigned to known VCGJ subgroups and did not form a unique group corresponding to their pathotype. ‘Soybean pathotype’ could not be distinguished among isolates of pathotype E by vegetative compatibility.     

Race Distribution, Vegetative Compatibility and Pathogenicity of Fusarium oxysporum f. sp. melonis Isolates in Greece

Races and vegetative compatibility groups (VCGs) in Greek isolates of Fusarium oxysporum f. sp. melonis(Fom) were characterized. Three races (0, 2 and 1–2) among 12 isolates tested and two VCGs among 19 isolates tested, were identified. Race 1–2 was the most common and race 1 was not detected. One widespread VCG corresponded to a VCG previously reported from Israel (coded 0138), and included seven isolates of races 0 and 1–2. The other VCG, which was unclassified, included four isolates of races 0, 2 and 1–2. The latter VCG was detected only in a specific melon‐growing location of Evros. The remaining eight isolates tested for VCG did not show positive reactions with other isolates, with each other or with the testers of VCGs 0135 or 0138, although they produced complementary mutants. Using two inoculation methods, the local cv. ‘Golden Head’ was found susceptible to all known Fom races, and especially to race 1–2. These results show the presence of more than one VCG and the widespread distribution of the race 1–2, in Greece.     

Genome-Wide Identification,Evolution and Expression Analyses of <i>GA2ox</i> Gene Family in <i>Brassica napus</i> L.

Gibberellin 2-oxidases (GA2ox) are important enzymes that maintain the balance of bioactive GAs in plants. GA2ox genes have been identified and characterized in many plants, but these genes were not investigated in Brassica napus. Here, we identified 31 GA2ox genes in B. napus and 15 of these BnaGA2ox genes were distributed in the A and C subgenomes. Subcellular localization predictions suggested that all BnaGA2ox proteins were localized in the cytoplasm, and gene structure analysis showed that the BnaGA2ox genes contained 2–4 exons. Phylogenetic analysis indicated that BnGA2ox family proteins in monocotyledons and dicotyledons can be divided into four groups, including two C₁₉-GA2ox and two C₂₀-GA2ox clades. Group 4 is a C₂₀-GA2ox Class discovered recently. Most BnaGA2ox genes had a syntenic relationship with AtGA2ox genes. BnaGA2ox genes in the C subgenome had experienced stronger selection pressure than genes in the A subgenome. BnaGA2ox genes were highly expressed in specific tissues such as those involved in growth and development, and most of them were mainly involved in abiotic responses, regulation of phytohormones and growth and development. Our study provided a valuable evolutionary analysis of GA2ox genes in monocotyledons and dicotyledons, as well as an insight into the biological functions of GA2ox family genes in B. napus.     

Triterpenoids from <i>Uncaria rhynchophylla</i> and Their PTP1B Inhibitory Activity

Uncaria rhynchophylla (Gouteng) is a famous traditional Chinese medicine used for psychiatric and hypotensive purposes in China. In this study, the ethyl acetate (EtOAc) part of U. rhynchophylla was revealed with protein tyrosine phosphatase 1B (PTP1B) inhibitory activity. Subsequent investigation on the EtOAc part yielded one new triterpenoid, 3β-formyloxy-6β,19α-dihydroxyurs-12-en-28-oic acid (1) and four known ones, 3β,6β,19α-trihydroxyurs-12-en-28-oic acid (2), 2-oxopomolic acid (3), 3β,19α-dihydroxy-6-oxo-olean-12-en-28-oic acid (4) and sumaresinolic acid (5). The structure of compound 1 was determined by extensive HRESIMS, IR, 1D and 2D NMR spectroscopic analyses. Two ursane-type triterpenoids (2 and 3) showed selective inhibition on PTP1B with IC₅₀ values of 48.2 and 178.7 μM. The enzyme kinetic study suggested that compounds 2 and 3 were mix-type inhibitors on PTP1B with K_i values of 15.6 and 132.5 μM. This investigation manifests the antidiabetic potency of U. rhynchophylla with triterpenoids as the active constituents.