Combined use of biocontrol agents and zeolite as a management strategy against <Emphasis Type="Italic">Fusarium</Emphasis> and <Emphasis Type="Italic">Verticillium</Emphasis> wilt

Vascular wilt pathogens, like Fusarium oxysporum and Verticillium dahliae, cause heavy economic loses to a range of crops. The lack of chemical control intensifies the problem. In the present study, the initial in vitro activity of 134 bacterial isolates, originating from various stages of the composting process of cotton residues, against F. oxysporum f. sp. melonis (FOM) and V. dahliae was evaluated. The most efficient strains, named SP10 and C20 M, belong to Bacillus sp. Both strains significantly reduced Fusarium and Vertilicillium wilt in melon and aubergine respectively. Furthermore, zeolite was tested alone or in combination with SP10 against V. dahliae and FOM. It was shown that the combination of zeolite and SP10 in the transplant soil plug was the most disease suppressive treatment. Interestingly the single application of zeolite was also plant-protective. The positive effect of zeolite on plant health could be linked with the recorded up-regulation of plant defense genes.     

Natural recovery from Verticillium wilt in olive: can it be exploited in a control strategy?

Aims

Our objective was to evaluate if natural recovery may be exploited in disease control of Verticillium wilt in olive. Therefore, we evaluated the following: the incidence of natural recovery; the Verticillium dahliae viability within olive tissues over time and the effectiveness of soil solarization, calcium cyanamide and pollarding of trees at soil level in promoting natural recovery.

Methods

Three different experiments (A, B and C) were performed in commercial olive orchards planted with the highly susceptible cv. ‘Bella di Cerignola’ and infested with the non-defoliating V. dahliae pathotype.

Results

In experiment A, in the period 2010–2012, natural recovery occurred on 35 of 138 diseased trees (25 %); however, this recovery was transient and lasted between 3 months for 11 trees (8 %) and 21 months for one tree (0.7 %). V. dahliae tended to be inactivated in twigs within 1 or 2 years after symptom onset (experiment A). However, it was evident that V. dahliae was more abundant in larger (trunk and first- or second-order branches) versus thinner woody parts of olive trees (roots; experiment B). In the attempt to explore whether natural recovery could be further stimulated artificially, it was observed that soil solarization and soil application of calcium cyanamide were ineffective in promoting its occurrence. Tree pollarding at soil level induced a transient recovery, which lasted only 1 year (experiment C).

Conclusions

Based on our observations, natural recovery of susceptible olive from Verticillium wilt has a low impact on the disease epidemiology in the short-term only and cannot be effectively stimulated in practice by soil solarization, calcium cyanamide or tree pollarding.     

MiR395 Overexpression Increases Eggplant Sensibility to <Emphasis Type="Italic">Verticillium dahliae</Emphasis> Infection

Verticillium wilt (V. wilt), a notorious wilt disease caused by Verticillium dahliae, often leads to the reduction of eggplant (Solanum melongena L.) production. MiRNAs, as a class of small RNAs, can regulate gene expression and then affect growth and development in plants. MiR395 has been proven to respond to sulfate-deficient stress in Arabidopsis thaliana and sulfate is well known to have a close relationship with plant disease resistance. To explore the function of eggplant miR395, we examined its expression in V. dahliae-infected eggplant by qRT-PCR and found miR395 exhibited a gradual reduction trend with time after infection. We then expressed pre-miR395 from Arabidopsis thaliana in Suqi eggplant and resistance analysis showed that miR395 overexpressed plants were hypersensitive to V. dahliae infection. We further measured the content of GSH and activities of POD and SOD and the results indicated that the index of GSH/POD/SOD in the overexpressed plants was lower than that of the wild-type control under V. dahliae infection. These results suggest that miR395 plays a negative role in eggplant response to V. dahliae infection.     

Genomic,evolutionary and expression profile analysis of <Emphasis Type="Italic">Hsp70</Emphasis> superfamily in A and D genome of cotton (<Emphasis Type="Italic">Gossypium</Emphasis> spp.) under the challenge of <Emphasis Type="Italic">Verticillium dahliae</Emphasis>

In this study, we comparatively analyzed the 115 Hsp70 genes identified in Gossypium raimondii, Gossypium hirsutum and Gossypium arboreum genomes. Those Hsp70 genes unequally distributed among chromosomes in A and D genome of cotton (Gossypium spp.), and were classified into 29 groups according to the homology of them. Based on the localization information of the orthologs in Arabidopsis, the Hsp70 proteins were predicted to locate in cytosol, endoplasmic reticulum, mitochondrion or chloroplast. Homologous analysis indicated the evolutionary conservation of Hsp70 in cotton. In addition, those Hsp70 genes were differently expressed in Suyuan-045, Hai-7124 and TM-1, which were highly resistant, resistant, and sensitive to Verticillium dahliae respectively. The expressions of 26 Hsp70 genes were induced by Verticillium dahliae except for Hsp70-07/16/25/26, and the result suggested the potential involvement of them in responding to Verticillium wilt. Hsp70-08/30/31 was highly expressed in both Suyuan-045 and Hai-7124, and it was hypothesized that they might be involved in the resistance to the invasion of Verticillium dahliae. 144h after inoculation with Verticillium dahliae, the expression of Hsp70-13/14/15 was only up-regulated in Suyuan-045, and it was assumed that they might be involved in resistance to the extension of Verticillium dahliae. Further study on those Hsp70 genes would be valuable to reveal the role of them in Verticillium wilt resistance.     

The purification and characterization of a novel hypersensitive-like response-inducing elicitor from Verticillium dahliae that induces resistance responses in tobacco

PevD1, a novel protein elicitor from the pathogenic cotton verticillium wilt fungus, Verticillium dahliae, induced a hypersensitive response in tobacco plants. In this paper, the elicitor was purified and analyzed using de novo sequencing. The protein-encoding pevD1 gene consists of a 468-bp open reading frame that produces a polypeptide of 155 amino acids, with a theoretical molecular weight of 16.23 kDa. The sequence of elicitor protein PevD1 was matched to the genomic sequence (GenBank accession no. ABJE 01000445.1) of a putative protein from V. dahliae strain vdls.17, but a function had not yet been reported. The pevD1 gene was expressed in Escherichia coli, and the recombinant protein was characterized for its ability to confer systemic acquired resistance to tobacco mosaic virus (TMV). Recombinant PevD1-treated plants exhibited enhanced systemic resistance compared to control, including a significant reduction in the number and size of TMV lesions on tobacco leaves. The elicitor protein-induced hydrogen peroxide production, extracellular-medium alkalization, callose deposition, phenolics metabolism, and lignin synthesis in tobacco. Our results demonstrate that elicitor-PevD1 triggers defense responses in intact tobacco plants.     

Silencing of <Emphasis Type="Italic">GbANS</Emphasis> reduces cotton resistance to <Emphasis Type="Italic">Verticillium dahliae</Emphasis> through decreased ROS scavenging during the pathogen invasion process

Anthocyanins are secondary metabolites that play important roles in plant adaption to adverse environments. The anthocyanin biosynthetic pathway is conserved in high plants. Previous studies revealed the significant role of anthocyanins in natural-colorized cotton. However, little is known about the involvement of anthocyanins in the interaction of cotton and pathogen. In this study, a pathogen-induced gene was isolated from Gossypium barbadense that encodes an anthocyanidin synthase protein (GbANS) with dioxygenase structures. GbANS was preferentially expressed in colored tissue. Silencing of GbANS significantly reduced the production of anthocyanins, as well as the cotton’s resistance to Verticillium dahliae. Biochemical studies revealed that GbANS-silenced cotton accumulated more hydrogen peroxide compared to control plants during the V. dahliae invasion process. This accumulation of hydrogen peroxide corresponded with increased cell death around the invasion sites, which in turn accelerated the V. dahliae infection. Taken together, we found that GbANS contributes to the biosynthesis of anthocyanins in cotton and anthocyanins positively regulate cotton’s resistance to V. dahliae.     

Ectopic expression of a novel Ser/Thr protein kinase from cotton (Gossypium barbadense), enhances resistance to Verticillium dahliae infection and oxidative stress in Arabidopsis

Key message

Overexpression of a cotton defense-related gene GbSTK in Arabidopsis resulted in enhancing pathogen infection and oxidative stress by activating multiple defense-signaling pathways.

Abstract

Serine/threonine protein kinase (STK) plays an important role in the plant stress-signaling transduction pathway via phosphorylation. Most studies about STK genes have been conducted with model species. However, their molecular and biochemical characterizations have not been thoroughly investigated in cotton. Here, we focused on one such member, GbSTK. RT-PCR indicated that it is induced not only by Verticillium dahliae Kleb., but also by signaling molecules. Subcellular localization showed that GbSTK is present in the cell membrane, cytoplasm, and nucleus. Overexpression of GbSTK in Arabidopsis resulted into the enhanced resistance to V. dahliae. Moreover, Overexpression of GbSTK elevated the expression of PR4, PR5, and EREBP, conferring on transgenic plants enhanced reactive oxygen species scavenging capacity and oxidative stress tolerance. Our results suggest that GbSTK is active in multiple defense-signaling pathways, including those involved in responses to pathogen infection and oxidative stress.     

The Thioredoxin GbNRX1 Plays a Crucial Role in Homeostasis of Apoplastic Reactive Oxygen Species in Response to Verticillium dahliae Infection in Cotton

Examining the proteins that plants secrete into the apoplast in response to pathogen attack provides crucial information for understanding the molecular mechanisms underlying plant innate immunity. In this study, we analyzed the changes in the root apoplast secretome of the Verticillium wilt-resistant island cotton cv Hai 7124 (Gossypium barbadense) upon infection with Verticillium dahliae. Two-dimensional differential gel electrophoresis and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry analysis identified 68 significantly altered spots, corresponding to 49 different proteins. Gene ontology annotation indicated that most of these proteins function in reactive oxygen species (ROS) metabolism and defense response. Of the ROS-related proteins identified, we further characterized a thioredoxin, GbNRX1, which increased in abundance in response to V. dahliae challenge, finding that GbNRX1 functions in apoplastic ROS scavenging after the ROS burst that occurs upon recognition of V. dahliae. Silencing of GbNRX1 resulted in defective dissipation of apoplastic ROS, which led to higher ROS accumulation in protoplasts. As a result, the GbNRX1-silenced plants showed reduced wilt resistance, indicating that the initial defense response in the root apoplast requires the antioxidant activity of GbNRX1. Together, our results demonstrate that apoplastic ROS generation and scavenging occur in tandem in response to pathogen attack; also, the rapid balancing of redox to maintain homeostasis after the ROS burst, which involves GbNRX1, is critical for the apoplastic immune response.Cotton (Gossypium spp.) is one of the most economically important crops worldwide and a number of pathogens affect the growth and development of cotton plants. The soil-borne pathogen Verticillium dahliae (V. dahliae) causes the destructive vascular disease Verticillium wilt, which results in devastating reductions in plant mass, lint yield, and fiber quality (Bolek et al., 2005; Cai et al., 2009). To date, Verticillium wilt has not been effectively controlled in the most common cultivated cotton species, upland cotton (Gossypium hirsutum), and cultivars with stably inherited resistance to this disease are currently unavailable (Aguado et al., 2008; Jiang et al., 2009; Zhang et al., 2012a). Unlike upland cotton, sea-island cotton (Gossypium barbadense), which is only cultivated on a small scale, possesses Verticillium wilt resistance. Exploring the molecular mechanisms involved in the defense responses against V. dahliae invasion in G. barbadense can provide useful information for generating wilt-resistant G. hirsutum species through molecular breeding.During the past decades, progress has been made in studying the defense responses against V. dahliae infection in cotton. Global analyses have demonstrated that several signaling pathways, including those mediated by salicylic acid, ethylene, jasmonic acid, and brassinosteroids, activate distinct processes involved in V. dahliae defense (Bari and Jones, 2009; Grant and Jones, 2009; Gao et al., 2013a). Accumulating evidence indicates that many V. dahliae-responsive genes, such as GbWARKY1, GhSSN, GbERF, GhMLP28, GhNDR1, GhMKK2, and GhBAK1 (Qin et al., 2004; Gao et al., 2011, 2013b; Li et al., 2014a; Sun et al., 2014; Yang et al., 2015), play crucial roles in defense against Verticillium wilt. In addition, the biosynthesis of terpenoids, lignin, and gossypol also makes important contributions to V. dahliae resistance in cotton (Tan et al., 2000; Luo et al., 2001; Xu et al., 2011; Gao et al., 2013a). Together, these studies have greatly improved our understanding of the complex innate defense systems against V. dahliae infection in cotton.The initial interaction between plants and pathogens takes place in the apoplast, the compartment of the plant cell outside the cell membrane, including the cell wall and intercellular space (Dietz, 1997). In response to pathogen colonization, the attacked plant cells undergo significant cellular and molecular changes, such as reinforcement of the cell wall and secretion of antimicrobial molecules into the apoplastic space (Bednarek et al., 2010). Thus, the apoplast serves as the first line of defense against microbe invasion, and apoplast immunity can be considered an important component of the plant immune response to pathogens.Upon recognition of pathogen infection, rapid production of reactive oxygen species [the reactive oxygen species (ROS) burst] occurs in the apoplast (Lamb and Dixon, 1997; Torres et al., 2006; Torres, 2010). This ROS burst is regarded as a core component of the early plant immune response (Daudi et al., 2012; Doehlemann and Hemetsberger, 2013). During defense responses, apoplastic ROS can diffuse into the cytoplasm and serve as signals, interacting with other signaling processes such as phosphorylation cascades, calcium signaling, and hormone-mediated pathways (Kovtun et al., 2000; Mou et al., 2003). Apoplastic ROS can also directly strengthen the host cell walls by oxidative cross linking of glycoproteins (Bradley et al., 1992; Lamb and Dixon, 1997) or the precursors of lignin and suberin polymers (Hückelhoven, 2007). Moreover, apoplastic ROS can directly affect pathogens by degrading nucleic acids and peptides from microbes or causing lipid peroxidation and membrane damage in the microbe (Mehdy, 1994; Lamb and Dixon, 1997; Apel and Hirt, 2004; Montillet et al., 2005).ROS levels in the apoplast increase rapidly in response to a variety of pathogens, but subsequently return to basal levels. The rapid production and dissipation of apoplastic ROS indicate that this process is finely regulated. Two classes of enzymes, NADPH oxidases and class III peroxidases, account for the rapid ROS burst in the apoplast (Bolwell et al., 1995; O’Brien et al., 2012). NADPH oxidases are directly phosphorylated by the receptor-like kinase BIK1 to enhance ROS generation (Li et al., 2014b). Also, due to the toxicity of high levels of ROS, plants have evolved enzymatic and nonenzymatic mechanisms to eliminate ROS, thereby preventing or reducing oxidative damage (Rahal et al., 2014; Torres et al., 2006). However, the molecular system responsible for the regulation of apoplastic ROS homeostasis during the immune response is not well understood.In this study, we performed a comparative analysis of the apoplastic proteomes in control roots compared with V. dahliae-inoculated roots of Gossypium barbadense (wilt-resistant sea-island cotton) using the two-dimensional differential gel electrophoresis (2D-DIGE) technique. Among the differentially expressed apoplastic proteins, ROS-related proteins were found to be major components, including a thioredoxin, GbNRX1, which functions as an ROS scavenger in response to V. dahliae infection. Knock-down of GbNRX1 expression in cotton by virus-induced gene silencing (VIGS) resulted in reduced resistance to V. dahliae. Our results demonstrate that maintaining apoplastic ROS homeostasis is a crucial component of the apoplastic immune response and that GbNRX1 is an important regulator of this process.     

Development and validation of a new real-time assay for the quantification of <Emphasis Type="Italic">Verticillium dahliae</Emphasis> in the soil: a comparison with conventional soil plating

Verticillium wilt of olive, caused by the soil-borne fungus Verticillium dahliae, is one of the most serious diseases of olive tree. In this study, a SYBR Green-based quantitative polymerase chain reaction (Q-PCR) assay targeting the intergenic spacer (IGS) region of the ribosomal DNA (rDNA) was developed to quantify V. dahliae microsclerotia (MS) in soils cropped with olive tree. In order to make the assay quantitative, the number of rDNA units in the genome was estimated using Q-PCR and fixed at 25 copies/genome. The assay was highly specific for V. dahliae, with no cross-amplification with other soil-borne pathogens. The sensitivity analysis showed similar slopes and efficiency, from both fungal DNA (slope?=??3.405, r²?=?0.976, E?=?96.64 %) and the positive recombinant plasmid (y?=??3.36, r²?=?0.989, E = 98.43 %), thus indicating a high accuracy of the assay. The assay exhibits a high intra- and inter-run reproducibility at a very low concentration of 10² copies/μL (CV%?≈?1 %). When the real-time PCR assay was applied to quantify MS in five naturally infested soil samples, it was able to detect V. dahliae in as few as two MS g^?1 of soil. Q-PCR estimates of pathogen DNA were significantly correlated with disease severity (r²?=?0.944) and with the soil plating method (r²?=?0.845). This new assay will be a valuable tool and can be applied for disease risk prediction before installing new plantations, and provides a more complete and rapid examination for soils subjected to such a treatment program.     

Expression of baculovirus anti-apoptotic genes p35 and op-iap in cotton (Gossypium hirsutum L.) enhances tolerance to verticillium wilt

Background

Programmed cell death plays an important role in mediating plant adaptive responses to the environment such as the invasion of pathogens. Verticillium wilt, caused by the necrotrophic pathogen Verticillium dahliae, is a serious vascular disease responsible for great economic losses to cotton, but the molecular mechanisms of verticillium disease and effective, safe methods of resistance to verticillium wilt remain unexplored.

Methodology/Principal Findings

In this study, we introduced baculovirus apoptosis inhibitor genes p35 and op-iap into the genome of cotton via Agrobacterium-mediated transformation and analyzed the response of transgenic plants to verticillium wilt. Results showed that p35 and op-iap constructs were stably integrated into the cotton genome, expressed in the transgenic lines, and inherited through the T₃ generation. The transgenic lines had significantly increased tolerance to verticillium wilt throughout the developmental stages. The disease index of T₁–T₃ generation was lower than 19, significantly (P<0.05) better than the negative control line z99668. After treatment with 250 mg/L VD-toxins for 36 hours, DNA from negative control leaves was fragmented, whereas fragmentation in the transgenic leaf DNA did not occur. The percentage of cell death in transgenic lines increased by 7.11% after 60 mg/L VD-toxin treatment, which was less than that of the negative control lines''s 21.27%. This indicates that p35 and op-iap gene expression partially protects cells from VD-toxin induced programmed cell death (PCD).

Conclusion/Significance

Verticillium dahliae can trigger plant cells to die through induction of a PCD mechanism involved in pathogenesis. This paper provides a potential strategy for engineering broad-spectrum necrotrophic disease resistance in plants.     

Introgression of bacterial wilt resistance from eggplant to potato via protoplast fusion and genome components of the hybrids

Key message

Bacterial wilt resistant somatic hybrids were obtained via protoplast fusion between potato and eggplant and three types of nuclear genomes were identified in the hybrids through GISH and SSR analysis.

Abstract

Cultivated potato (Solanum tuberosum L.) lacks resistance to bacterial wilt caused by Ralstonia solanacearum. Interspecific symmetric protoplast fusion was conducted to transfer bacterial wilt resistance from eggplant (S. melongena, 2n = 2x = 24) into dihaploid potato (2n = 2x = 24). In total, 34 somatic hybrids were obtained, and of these, 11 rooted and were tested for genome components and resistance to race 1 of R. solanacearum. The hybrids exhibited multiple ploidy levels and contained the dominant nuclear genome from the potato parent. Three types of nuclear genomes were identified in the hybrids through genomic in situ hybridization (GISH) and simple sequence repeat (SSR) analysis, including (1) the potato type of the tetraploids in which eggplant chromosomes could not be detected by GISH but their nuclear DNA was confirmed by SSR, (2) the biased type of the hexaploids in which the chromosome dosage was 2 potato:1 eggplant, and (3) the chromosome translocation type of the mixoploids and aneuploids that was characterized by various rates of translocations of nonhomologous chromosomes. Cytoplasmic genome analysis revealed that mitochondrial DNA of both parents coexisted and/or recombined in most of the hybrids. However, only potato chloroplast DNA was retained in the hybrids speculating a compatibility between cpDNA and nuclear genome of the cell. The pathogen inoculation assay suggested a successful transfer of bacterial wilt resistance from eggplant to the hybrids that provides potential resistance for potato breeding against bacterial wilt. The genome components characterized in present research may explain partially the inheritance behavior of the hybrids which is informative for potato improvement.