The role of the β-1,6-endoglucanase gene vegB in physiology and virulence of Verticillium dahliae

The β-1,6-endoglucanase gene (vegB) of Verticillium dahliae was isolated using a genome walking technique. Nucleotide and deduced amino acid sequences of the gene showed high identity with the PAN1 sequence deposited at the Verticillium genome database (Broad Institute), but significant differences in intron numbers and sites of insertion. Detailed in silico analysis, accompanied by sequencing of both genomic and cDNA, as well as RT-PCR experiments, provided the correct size of the gene and the exact number, length and positions of introns. The putative protein of this gene was compared with corresponding β-1,6-endoglucanases from other fungi, and sequences were used to construct a phylogenetic tree. A clear differentiation between enzymes derived from plant pathogenic and mycoparasitic fungi was observed, fully supported by bootstrap data. An internal fragment (1.2kb) of vegB was used to disrupt the wild-type gene of a V. dahliae tomato race 2 strain, and the mutant strain, vegB-, was tested for pathogenicity on tomato plants. Results showed a small but constant reduction in disease symptoms only on eggplants for the vegB- strain in comparison with the wild type. Growth on minimal medium supplemented with different carbon sources showed reduced ability of the mutant to breakdown cellulose, whereas growth on glucose, pectin and sucrose was similar to the wild type

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

Ceratocystis ficicola causing a serious disease of Ficus carica in Greece

Ceratocystis ficicola causes vascular wilt of fig trees in Japan, invading root systems and the main stems eventually leading to tree death. In surveys from 2018 to 2020 in fig orchards in Greece, this fungus was detected in two separated regions. The fungus was consistently isolated from infected wood and from rhizosphere soil. The isolates were identified based on multi-locus phylogenetic analyses of rpb2, bt1 and tef1 gene regions and detailed morphological characteristics, including comparisons with an ex-type isolate of C. ficicola from Japan. The pathogenicity of Greek isolates was proven on Ficus carica and F. benjamina plants. Ceratocystis ficicola is a soilborne pathogen, and the occurrence of vascular wilt outbreaks suggest that the pathogen spreads within and between orchards with infested soil and wood debris during ploughing. The pathogen is also spreading in Greece with infected propagation material. This is the first detailed report of C. ficicola outside Japan, and there is concern over potential spread of the pathogen to other Mediterranean countries, where approx. 70% of the world fig production occurs.The Department of Science and Technology/National Research Foundation Center of Excellence in Plant Health Biotechnology, South Africa.https://oajournals.fupress.net/index.php/pmam2022BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog

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

Crosstalk between the cAMP-PKA pathway and the beta-1,6-endoglucanase in Verticillium dahliae

In plant pathogenic fungi, different signalling pathways operate to control responses to nutrient availability during plant infection. A candidate from the cAMP-PKA signalling pathway, the cAMP-dependent protein kinase A gene, pkaC1, and the beta-1,6-endoglucanase gene, vegB, involved in cell wall degradation, were studied in V. dahliae. Double mutants of the fungus were constructed, with insertional inactivation in the pkaC1 and vegB genes. Different developmental traits and virulence towards eggplant were evaluated in single and double disruption mutants. In all media tested, double mutants showed better radial growth but less conidia and microsclerotia than the wild type. An interaction between vegB and pkaC1 in controlling virulence on eggplants was recorded, as double mutants were slightly less virulent than the single mutant vegB(-), but more virulent than the single mutant pkaC1(-). Concomitant or independent function of the two genes and the signaling pathways they operate in for the different growth parameters and virulence are discussed

Thermal inactivation of compost suppressiveness implicates possible biological factors in disease management

Verticillium wilt is a devastating disease of a wide range of herbaceous and woody plant hosts, incited by the soilborne fungus Verticillium dahliae. Since there are no chemical treatments to control the pathogen, management strategies are focused on preventive measures. In the present study, the microbial nature involved in the suppressiveness of a compost amendment (GR6, fabricated of horse manure, unbroken bedding hay + wood shavings and municipal green waste) against V. dahliae was investigated. For this purpose, eggplants grown in sterilised or non-sterilised compost were transplanted in soil infested with V. dahliae microsclerotia, amended or not with sterilised or non-sterilised compost. The most efficient treatments were those that included non-sterilised compost; therefore, the observed suppressiveness could be attributed to microbial agents. Several microbes were isolated from the root system of eggplants grown in the compost and tested in vitro against V. dahliae. Two bacterial strains identified as members of the Pseudomonas fluorescens complex and two fungal isolates belonging to Fusarium oxysporum were selected for further evaluation under glasshouse conditions. The ability of the microbial agents to reduce the percentage of diseased leaves compared to the control treatment was demonstrated. Moreover, it was shown that the most efficient bacterial strain was a rhizosphere and endophyte inhabitant. In a field experiment, the treatment where compost was applied both in the nursery and the field and treatments with F. oxysporum and P. fluorescens strains exhibited reduced disease severity; however, an increase in yield compared to the untreated control was not observe

Evaluation of application methods and biocontrol efficacy of paenibacillus alvei strain K-165, against the cotton black root rot pathogen thielaviopsis basicola

Due to limited means for control, the black root rot fungus, Thielaviopsis basicola constitutes a major threat to organic and conventional cotton production worldwide. Despite the substantial yield loss and reduced fiber quality caused by the fungus, little attention has been drawn to the biological control of black root rot disease. In the present study, the efficacy of the biocontrol agent K-165 to control T. basicola on cotton was evaluated. Paenibacillus alvei strain K-165 is a biocontrol agent that has been studied extensively in the past against the soilborne pathogen Verticillium dahliae. K-165 inhibited T. basicola growth in vitro through antibiosis and reduced significantly root discoloration and hypocotyl lesions on cotton seedlings compared to the control treatment, under greenhouse conditions. In the in planta experiments three different application methods of the biocontrol agent were tested. It was shown that a seed coating treatment consisting of K-165 xanthan gum and talc was the most effective in reducing disease symptoms and increasing plant height and fresh weight compared to two other treatments (seed coating or pellets) based on sodium alginate. The efficacy of the K-165 xanthan gum-talc treatment was attributed to the higher bacterial concentration delivered to the seed and the subsequent higher bacterial population on the rhizosphere and soil compared to the other two application methods. K-165 was proven to be an efficient root and soil colonizer inhibiting extensive root colonization by T. basicola as it was shown by qPCR analysi

The ethylene receptor ETR1 is required for Fusarium oxysporum pathogenicity

Fusarium oxysporum is a ubiquitous vascular wilt plant pathogen causing severe yield losses in a wide range of economically important crops. In this study, the interaction between Fusarium oxysporum f. sp. raphani and Arabidopsis thaliana plants impaired in the salicylate (SA), jasmonate (JA) and ethylene (ET) defence signalling pathways was investigated to better understand the nature of this plant-microbe interaction. The in planta bioassays revealed a key role for the ETR1 receptor as the etr1-1 mutant plants exhibited statistically less Fusarium wilt symptoms compared to the other mutant and Col-0 plants. Quantitative polymerase chain reaction (qPCR) analysis associated the decrease in symptom severity shown in etr1-1 plants with reduced vascular growth of the pathogen, suggesting the activation of defence mechanisms in etr1-1 plants against F. oxysporum. Furthermore, the early activation and increased accumulation of the SA-responsive PR1, PR2 and PR5 genes in the etr1-1 plants, in contrast to the Col-0 plants that showed higher transcript levels of the JA/ET-responsive PR3, PR4 and PDF1.2 genes after F. oxysporum inoculation, can lead to speculation that F. oxysporum hijacks ETR1-mediated ethylene signalling to promote disease development in plants

Combined Transcriptomic and Metabolomic Analysis Reveals Insights into Resistance of Arabidopsis <i>bam3</i> Mutant against the Phytopathogenic Fungus <i>Fusarium oxysporum</i>

The wilt-inducing strains of Fusarium oxysporum are responsible for severe damage to many economically important plant species. The most cost-effective and environmentally safe method for the management of Fusarium wilt is the use of resistant cultivars when they are available. In the present study, the Arabidopsis genotype with disruptions in the β-amylase 3 (BAM3) gene, which encodes the major hydrolytic enzyme that degrades starch to maltose, had significantly lower susceptibility to Fusarium oxysporum f. sp. raphani (For) compared to wild-type (wt) plants. It showed the lowest disease severity and contained reduced quantities of fungal DNA in the plant vascular tissues when analyzed with real-time PCR. Through metabolomic analysis using gas chromatography (GC)–mass spectrometry (MS) and gene-expression analysis by reverse-transcription quantitative PCR (RT-qPCR), we observed that defense responses of Arabidopsis bam3 mutants are associated with starch-degradation enzymes, the corresponding modification of the carbohydrate balance, and alterations in sugar (glucose, sucrose, trehalose, and myo-inositol) and auxin metabolism