The innate immune signaling system as a regulator of disease resistance and induced systemic resistance activity against Verticillium dahliae

In the last decades, the plant innate immune responses against pathogens have been extensively studied, while biocontrol interactions between soilborne fungal pathogens and their hosts have received much less attention. Treatment of Arabidopsis thaliana with the nonpathogenic bacterium Paenibacillus alvei K165 was shown previously to protect against Verticillium dahliae by triggering induced systemic resistance (ISR). In the present study, we evaluated the involvement of the innate immune response in the K165-mediated protection of Arabidopsis against V. dahliae. Tests with Arabidopsis mutants impaired in several regulators of the early steps of the innate immune responses, including fls2, efr-1, bak1-4, mpk3, mpk6, wrky22, and wrky29 showed that FLS2 and WRKY22 have a central role in the K165-triggered ISR, while EFR1, MPK3, and MPK6 are possible susceptibility factors for V. dahliae and bak1 shows a tolerance phenomenon. The resistance induced by strain K165 is dependent on both salicylate and jasmonate-dependent defense pathways, as evidenced by an increased transient accumulation of PR1 and PDF1.2 transcripts in the aerial parts of infected plants treated with strain K165

Pyricularia leaf spot: A new disease of ornamental plants of the family Marantaceae

A severe leaf spot disease incited by the fungus Pyricularia oryzae was identified on Ctenanthe oppenheimiana and C. setosa "Greystar." Primary symptoms on young leaves consisted of individual circular to slightly irregular pinpoint spots with white necrotic centers zonated by narrow brown-yellow halos. On mature leaves, extended necrotic areas resembling those caused by phytotoxicity were formed. Artificially inoculated leaves with a spore suspension of the isolated fungus from the above case showed symptoms after 3 to 4 days' incubation at 25 degrees C and high humidity. Fungal isolates obtained from Ctenanthe plants of Brazilian origin were found to be highly pathogenic on various plants within the family Marantaceae when they were tested by an excised leaf assay method. By contrast, P. oryzae isolates obtained from rice plants grown in Greece caused either hypersensitivity or immune response symptoms in various Marantaceae. Analysis of esterase and lactate dehydrogenase isozymes showed different banding patterns for rice and Ctenanthe isolates of P. oryzae. Conditions of prolonged leaf wetness combined with prevailing high temperature and humidity favored the epidemic appearance of the Pyricularia leaf spot disease on glasshouse-grown plants during the summer months of 1995 in Greece

A Relationship among fungicide-resistant phenotypes of Botrytis cinerea based on RAPD analysis

On the basis of the mode of spore germination and mycelial growth on fungicide-amended media, 200 Botrytis cinerea Pers.:Fr. single spore isolates were classified into six representative groups of resistant phenotypes. Sixty eight isolates were phenylcarbamate highly resistant (PcmHR, wild type), eight were dicarboximide moderately and phenylcarbamate highly resistant (DicMRPCMHR), four were benzimidazole and phenylcarbamate highly resistant (BenHRPcmHR), five were dicarboximide and benzimidazole moderately resistant and phenylcarbamate highly resistant (DicMR BenMRPcmHR), twenty were dicarboximide moderately and benzimidazole highly resistant (DicMRBenHR) and ninety five isolates were benzimidazole highly resistant (BEN HR). Two representative isolates of each phenotype were examined by random amplified polymorphic DNA (RAPD) fingerprint analysis. Relationships among the isolates were determined using the unweighted pair-group method with arithmetic average (UP-GMA) and dendrograms were constructed. Regardless of host, geographical origin and year of the isolation, phylogenetic analysis revealed the clear differentiation of the six phenotypic-resistant groups classified into three clusters. The first cluster included isolates of the PcmHR phenotype with subdivisions included the wild type, the DicMR and the BenHR phenotypes. The latter two phenotypes maintained their wild type insensitivity to diethofencarb. This first cluster was branching with a second cluster that included the double resistance phenotypes DicMRBenMR and DicMRBen HR. A third cluster, which was most distantly related to the above two included isolates of BenHR only. The presented data show a positive correlation between conventional and molecular techniques in definition of fungicide-resistant phenotypes and support earlier findings of the genetically based diversity of fungicide resistance

Ethylene perception via ETR1 is required in Arabidopsis infection by Verticillium dahliae

Vascular wilts caused by Verticillium spp. are very difficult to control and, as a result, are the cause of severe yield losses in a wide range of economically important crops. The responses of Arabidopsis thaliana mutant plants impaired in known pathogen response pathways were used to explore the components in defence against Verticillium dahliae. Analysis of the mutant responses revealed enhanced resistance in etr1-1 [ethylene (ET) receptor mutant] plants, but not in salicylic acid-, jasmonic acid- or other ET-deficient mutants, indicating a crucial role of ETR1 in defence against this pathogen. Quantitative polymerase chain reaction analysis revealed that the decrease in symptom severity shown in etr1-1 plants was associated with significant reductions in the growth of the pathogen in the vascular tissues of the plants, suggesting that impaired perception of ET via ETR1 results in increased disease resistance. Furthermore, the activation and increased accumulation of the PR-1, PR-2, PR-5, GSTF12, GSTU16, CHI-1, CHI-2 and Myb75 genes, observed in etr1-1 plants after V. dahliae inoculation, indicate that the outcome of the induced defence response of etr1-1 plants seems to be dependent on a set of defence genes activated on pathogen attac

Insights into the role of ethylene perception in tomato resistance to vascular infection by Verticillium dahliae

A Tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) system was employed to investigate the role of the tomato ethylene receptor ETR4. By comparing wilting symptoms of verticillium wilt in wild-type, ethylene-insensitive Never ripe (Nr) mutant tomato plants and ETR4-silenced plants, it was demonstrated that disease severity in the Nr and ETR4-silenced plants was statistically reduced compared to wild-type plants. Disease incidence and severity were reduced by 11 and 20%, respectively, in the Nr plants compared to the wild-type plants, at 33 days post-inoculation (d.p.i.). In the ETR4-silenced plants, disease incidence and severity were reduced by 14 and 15%, respectively, compared to the TRV-only-inoculated plants, at 37 d.p.i. Quantification of Verticillium dahliae by qPCR revealed that the reduction in symptom severity in the Nr plants was associated with significant reduction of growth of the pathogen in the vascular tissues of the Nr plants compared to that in the wild-type plants, suggesting that impaired perception of ethylene via the Never-ripe receptor results in increased disease resistance. Fungal reduction was evident at each sampling day in the Nr plants, ranging from 1·5 to 1·75 times less than that in the wild-type plants. Fungal quantification in the ETR4-silenced and TRV-only-inoculated plants showed similar levels of fungal biomas

Transformation and attachment of Beauveria bassiana conidia on the cuticle of Tribolium confusum and Sitophilus oryzae in conjunction with diatomaceous earth

Agrobacterium tumefaciens-mediated transformation (AMT) was used in order to create fluorescent conidia of Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales). From the fungal transformants, one Enhanced Green Fluorescent Protein B. bassiana strain indicated stability (maintained the GFP characteristics) in fluorescent signal, while its pathogenicity did not differ significantly from the wild B. bassiana type. After exposure on wheat treated with the transformed conidia, fluorescence was evident on all body parts but especially on metasternon of adults of Tribolium confusum Jacquelin du Val (Coleoptera: Tenebrionidae) and on elytra in of Sitophilus oryzae (L.) (Coleoptera: Curculionidae). No significant differences were noted in fluorescence levels in the case of T. confusum between the transformed and the wild strain. However, the addition of diatomaceous earth (DE) increased attachment, especially at increased intervals after exposure. For S. oryzae, fluorescence of the transformed strain was decreased with the increased post-exposure time, but the simultaneous presence of DE increased fluorescence. The results of this study demonstrate that fluorescent conidia of B. bassiana can be utilized as a tool for the evaluation of parameters that affect the attachment of entomopathogenic fungi in insects' cuticle

Cloning biologically active geminivirus DNA using PCR and overlapping primers.

We report a new strategy for cloning DNA of the plant-infecting geminiviruses that is based on PCR amplification with primers overlapping the sequence of a restriction site in the viral genome. This method reduces the time and work necessary to obtain biologically active geminivirus DNAs and could be used for other DNA viruses having circular genomes, such as the animal-infecting circoviruses. Geminiviruses possess circular single-stranded DNA genomes that are monopartite [one DNA of ca. 2.6 kilobases (kb)] or bipartite (two DNAs designated DNA A and DNA B, each ca. 2.6 kb) and are encapsidated within twinned icosahedral particles (1). Replication of the viral genome occurs within plant cell nuclei via circular double-stranded replicative form (RF) DNA. RF DNA has been used to obtain infectious clones of several geminiviruses (2, 3, 4). Typically, RF DNA is isolated from infected plant tissue, a unique restriction site i