Sotirios E. Tjamos

Induction of Resistance to Verticillium dahliae in Arabidopsis thaliana by the Biocontrol Agent K-165 and Pathogenesis-Related Proteins Gene Expression

The biocontrol bacterium Paenibacillus alvei K165 has the ability to protect Arabidopsis thaliana against Verticillium dahliae. A direct antagonistic action of strain K165 against V. dahliae was ruled out, making it likely that K165-mediated protection results from induced systemic resistance (ISR) in the host. K165-mediated protection was tested in various Arabidopsis mutants and transgenic plants impaired in defense signaling pathways, including NahG (transgenic line degrading salicylic acid [SA]), etr1-1 (insensitive to ethylene), jar1-1 (insensitive to jasmonate), npr1-1 (nonexpressing NPR1 protein), pad3-1 (phytoalexin deficient), pad4-1 (phytoalexin deficient), eds5/sid1 (enhanced disease susceptibility), and sid2 (SA-induction deficient). ISR was blocked in Arabidopsis mutants npr1-1, eds5/sid1, and sid2, indicating that components of the pathway from isochorismate and a functional NPR1 play a crucial role in the K165-mediated ISR. Furthermore, the concomitant activation and increased transient accumulation of the PR-1, PR-2, and PR-5 genes were observed in the treatment in which both the inducing bacterial strain and the challenging pathogen were present in the rhizosphere of the A. thaliana plants.

Selection and screening of endorhizosphere bacteria from solarized soils as biocontrol agents against Verticillium dahliae of solanaceous hosts

Verticillium dahliae antagonistic endorhizosphere bacteria were selected from root tips of tomato plants grown in solarized soils. Fifty-three out of the 435 selected bacterial isolates were found to be antagonistic against V. dahliae and several other soilborne pathogens in dual cultures. Significant biocontrol activity against V. dahliae in glasshouse trials was demonstrated in three of 18 evaluated antagonistic isolates, provisionally identified as Bacillus sp. Although fluorescent pseudomonads were also isolated from root tips of tomato plants, none of the tested isolates exercised any significant antagonistic activity against V. dahliae in dual cultures. So these isolates were not tested in glasshouse trials in this study. Finally, two of the most effective bacterial isolates, designated as K-165 and 5-127, were shown to be rhizosphere colonizers, very efficient in inhibiting mycelial growth of V. dahliae in dual cultures and successfully controlling Verticillium wilt of solanaceous hosts. In glasshouse experiments, root dipping or soil drenching of eggplants with bacterial suspension of 107cfu ml−1 resulted in reduced disease severity expressed as percentage of diseased leaves (40–70%) compared to the untreated controls under high V. dahliae inoculum level (40 microsclerotia g−1 soil). In heavily Verticillium infested potato fields, experiments with potato seeds dusted with a bacterial talc formulation (108cfu g−1 formulation), showed a significant reduction in symptom development expressed as percentage of diseased potato plants and a 25% increase in yield over the untreated controls. As for their effectiveness in increasing plant height, both bacterial isolates K-165 and 5-127 produced indolebutyric, indolepyruvic and indole propionic acids. Both antagonists are considered as plant growth promoting rhizobacteria bacteria since significantly increased the height of treated plants compared with the untreated controls. Chitinolytic activity test showed that both isolates were able to produce chitinase. Testing rhizospheric and endophytic activity of the antagonists it was shown that although the bacteria are rhizosphere inhabitants they also preferentially colonize the endorhizosphere of tomatoes and eggplants. Fatty acid analysis showed that isolate K-165 could belong to Paenibacillus alvei while 5-127 to Bacillus amiloliquefaciens.

Reactive oxygen species and plant resistance to fungal pathogens.

Reactive oxygen species (ROS) have been studied for their role in plant development as well as in plant immunity. ROS were consistently observed to accumulate in the plant after the perception of pathogens and microbes and over the years, ROS were postulated to be an integral part of the defence response of the plant. In this article we will focus on recent findings about ROS involved in the interaction of plants with pathogenic fungi. We will describe the ways to detect ROS, their modes of action and their importance in relation to resistance to fungal pathogens. In addition we include some results from works focussing on the fungal interactor and from studies investigating roots during pathogen attack.

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 attack.

Symptom development, pathogen isolation and Real-Time QPCR quantification as factors for evaluating the resistance of olive cultivars to Verticillium pathotypes

Verticillium wilt is the most serious olive disease in the Mediterranean countries and worldwide. The most effective control strategy is the use of resistant cultivars. However, limited information is available about the level and source of resistance in most of the olive cultivars and there are no published data using microsclerotia, the resting structures of Verticillium dahliae, as the infective inoculum. In the present study, we correlated symptomatology and the presence of the fungus along with the DNA relative amount (molecules μl−1) of a defoliating (D) and a non-defoliating (ND) V. dahliae strain in the susceptible cv. Amfissis and the tolerant cvs Kalamon and Koroneiki, as quantified by the Real-Time QPCR technology. The viability of the pathogen in the plant tissues was confirmed by isolating the fungus on PDA plates, while symptom assessment proved the correlation between the DNA relative amount of V. dahliae in plant tissues and cultivar susceptibility. It was further demonstrated that the D and ND strains were present at a significantly higher level in cv. Amfissis than in cvs Kalamon and Koroneiki. It was finally observed that the relative amount of the pathogen in roots was lower than in stems and shoots and declined in plant tissues over time. These data constitute a valuable contribution in evaluating resistance of olive cultivars or olive root-stocks to V. dahliae pathotypes.

Phenolic Responses of Resistant and Susceptible Olive Cultivars Induced by Defoliating and Nondefoliating verticillium dahliae Pathotypes

Verticillium wilt is the most serious olive disease worldwide. The olive-infecting Verticillium dahliae pathotypes have been classified as defoliating (D) and nondefoliating (ND), and the disease is mainly controlled in olive orchards by using resistant or tolerant cultivars. Limited information is available about the nature of resistance in most of the olive cultivars. In the present study, the phenolic responses of the susceptible to V. dahliae olive cv. Amfissis and the resistant cv. Koroneiki upon D and ND V. dahliae infection were monitored in relation to the fungal DNA levels in the vascular tissues with the purpose to explore the defense mechanisms of olive trees against V. dahliae. Quantitative polymerase chain reaction revealed that the decrease in symptom severity shown in Koroneiki trees was associated with significant reduction in the growth of both V. dahliae pathotypes in the vascular tissues compared with Amfissis. In Koroneiki trees, the levels of o-diphenols and verbascoside were positively associated with the DNA levels of the D and ND pathotypes. In addition, a positive association was observed between the levels of verbascoside and the fungal DNA level in Amfissis trees, whereas a negative association was revealed between the fungal DNA level and the total phenols and oleuropein content in both cultivars. The levels of verbascoside were clearly higher in Koroneiki trees compared with Amfissis trees, indicating for the first time in the literature the involvement of verbascoside in the defense mechanism of olive trees against V. dahliae.

Evaluation of compost amendments for suppressiveness against Verticillium wilt of eggplant and study of mode of action using a novel Arabidopsis pathosystem

The induced resistance potential of eleven compost samples that originated from four different countries (Greece, France, Netherlands and Israel) and were manufactured from various raw materials, was evaluated in an Arabidopsis thaliana–Verticillium dahliae pathosystem under greenhouse conditions using a novel Plexiglas chamber. Five out of eleven composts tested showed significant disease suppressiveness compared to the control treatment; three composts exhibited disease severity equal to the control, while in the other three composts, disease severity was higher than the control treatment. Two of the tested composts that showed strong or medium suppressiveness were further evaluated under field conditions against Verticillium wilt of eggplant. Neither of them significantly reduced disease severity or resulted in higher fruit yield in a semi-commercial field test although they could induce a systemic resistance response in the greenhouse. However, as a consequence of a growth-promoting effect, one of the compost samples tested in the field resulted in a significant yield increase compared with the other.

Implementation of soil solarization in Greece: conclusions and suggestions

Abstract The use of soil solarization in Greece is described, indicating methods of reducing the period of solarization by using impermeable plastic sheets and combining it with the addition of antagonistic bacteria.

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.

Biological control of Pythium, Rhizoctonia and Sclerotinia in lettuce: association of the plant protective activity of the bacterium Paenibacillus alvei K165 with the induction of systemic resistance

The soilborne fungi Sclerotinia sclerotiorum, Rhizoctonia solani and the oomycete Pythium ultimum are among the most destructive pathogens for lettuce production. The application of the biocontrol agent Paenibacillus alvei K165 to the transplant soil plug of lettuce resulted in reduced S. sclerotiorum, R. solani and P. ultimum foliar symptoms and incidence compared to untreated controls, despite the suppressive effect of the pathogens on the rhizosphere population of K165. In vitro, K165 inhibited the growth of S. sclerotiorum and R. solani but not P. ultimum. Furthermore, the expression of the pathogenesis-related (PR) gene PR1, a marker gene of salicylic acid (SA)-dependent plant defence, and of the Lipoxygenase (LOX) and Ethylene response factor 1 (ERF1) genes, markers of ethylene/jasmonate (ET/JA)-dependent plant defence was recorded. K165-treated plants challenged with P. ultimum showed up-regulation of PR1, whereas challenge with R. solani resulted in up-regulation of LOX and ERF1, and challenge with S. sclerotiorum resulted in up-regulation of PR1, LOX and ERF1. This suggests that K165 triggers the SA- and the ET/JA-mediated induced systemic resistance against P. ultimum and R. solani, respectively, while the simultaneous activation of the SA and ET/JA signalling pathways is proposed for S. sclerotiorum.