The volatile-producing Flavobacterium johnsoniae strain GSE09 shows biocontrol activity against Phytophthora capsici in pepper

Aims: Previously, we selected a bacterial strain (GSE09) antagonistic to Phytophthora capsici on pepper, which produced a volatile compound (2,4‐di‐tert‐butylphenol), inhibiting the pathogen. In this study, we identified strain GSE09 and characterized some of the biological traits of this strain in relation to its antagonistic properties against P. capsici. In addition, we examined bacterial colonization on the root surface or in rhizosphere soil and the effect of various concentrations of the volatile compound and strain GSE09 on pathogen development and radicle infection as well as radicle growth. Methods and Results: Strain GSE09 was identified as Flavobacterium johnsoniae, which forms biofilms and produces indolic compounds and biosurfactant but not hydrogen cyanide (HCN) with little or low levels of antifungal activity and swimming and swarming activities. Fl. johnsoniae GSE09 effectively colonized on pepper root, rhizosphere, and bulk (pot) soil, which reduced the pathogen colonization in the roots and disease severity in the plants. Various concentrations of 2,4‐di‐tert‐butylphenol or strain GSE09 inhibited pathogen development (mycelial growth, sporulation, and zoospore germination) in I‐plate (a plastic plate containing a center partition). In addition, germinated seeds treated with the compound (1–100 μg ml^?1) or the strain (10²–10¹⁰ cells ml^?1) significantly reduced radicle infection by P. capsici without radicle growth inhibition. Conclusions: These results indicate that colonization of pepper root and rhizosphere by the Fl. johnsoniae strain GSE09, which can form biofilms and produce indolic compounds, biosurfactant, and 2,4‐di‐tert‐butylphenol, might provide effective biocontrol activity against P. capsici. Significance and Impact of the Study: To our knowledge, this is the first study demonstrating that the Fl. johnsoniae strain GSE09, as a potential biocontrol agent, can effectively protect pepper plants against P. capsici infection by colonizing the roots.     

Optimization of Culturing Conditions of a Strain of Phytophthora capsici Pathogenic to Habanero Pepper (Capsicum chinense)

Phytophthora capsici is an oomycete known as the causal agent of wilting disease in Capsicum spp., which causes rotting of roots, crowns, stems, leaves and fruits. To date, little is known about the production of phytotoxic metabolites by P. capsici or their role in the infection process. As part of a project directed towards the isolation and identification of phytotoxins produced by a strain of P. capsici pathogenic to habanero pepper (Capsicum chinense), we have evaluated the effect of factors such as aeration, light and culture medium on the production of mycelium and phytotoxic metabolites by P. capsici. The results showed that culturing P. capsici in potato dextrose broth (PDB) containing habanero pepper leaf infusion, in the dark and under still conditions, results in a high production of mycelium and a high phytotoxicity of the culture filtrate, in the shortest period of time.     

Characterization of antifungal activity of Paenibacillus ehimensis KWN38 against soilborne phytopathogenic fungi belonging to various taxonomic groups

Soilborne fungal phytopathogens cause significant losses in many economically important crops and vegetables. The only way to control these devastating pathogens is by using higher doses of fungicides which not only increase the cost of production but also cause significant damage to the environment. Therefore alternate control measures are always looked for. In the present study, an antagonistic strain was isolated from the soil of the pepper fields around the seashore of Jellanamdo, South Korea and identified as Paenibacillus ehimensis KWN38 based on 16S rRNA sequencing. The strain showed high antifungal activity against six tested fungal pathogens belonging to various taxonomic groups on dual culture plates. Furthermore, the strain produced volatile antimicrobial compounds which had strong fungal growth inhibitory effect. The strain also showed high chitinase, cellulase, glucanase and protease activities. The hyphal morphologies of Rhizoctonia solani AG-1 (IA), Fusarium oxysporum f.sp. lycopersici and Phytophthora capsici were significantly destroyed by the crude enzymes and butanol extract from the culture supernatant and the affected hyphae showed abnormal bending, tip curling, and irregular branching. Hence, Paenibacillus ehimensis KWN38 is considered as a potential biocontrol agent of the soil-borne fungi causing plant diseases which is an important perspective of the present study.     

In vitro and in vivo antimicrobial activity of Xenorhabdus bovienii YL002 against Phytophthora capsici and Botrytis cinerea

Aims: Developing new bio‐agents to control plant disease is desirable. Entomopathogenic bacteria Xenorhabdus spp. have potential antimicrobial activity in agriculture. This work was conducted to evaluate the antimicrobial activity of Xenorhabdus bovienii YL002 on plant pathogenic fungi and oomycete in vitro and the efficiency of this strain to reduce the in vivo incidence of grey mould rot on tomato plants caused by Botrytis cinerea and leaf scorch on pepper plants caused by Phytophthora capsici. Methods and Results: The antimicrobial activity of X. bovienii YL002 was firstly determined on in vitro plant pathogenic fungi and oomycete and then on tomato fruits and plants infected with B. cinerea and pepper plants infected with P. capsici. The cell‐free filtrate of X. bovienii YL002 exhibited highest inhibition effects (>98%) on mycelia growth of P. capsici and B. cinerea. The 50% inhibition concentration (EC₅₀) of the methanol‐extracted bioactive compounds (methanol extract) of the cell‐free filtrate against P. capsici and B. cinerea were 164·83 and 42·16 μg ml^?1. The methanol extract also had a strong effect on the spore germination of P. capsici and B. cinerea, with a EC₅₀ of 70·38 and 69·33 μg ml^?1, respectively. At 1000 μg ml^?1, the methanol extract showed a therapeutic effect of 70·82% and a protective effect of 77·4% against B. cinerea on tomato plants compared with the control. The methanol extract also showed potent effect against P. capsici, with a therapeutic effect of 68·14% and a protective effect of 65·46% on pepper plants compared with the control. Conclusions: Xenorhabdus bovienii YL002 produces antimicrobial compounds with strong activity on plant pathogenic fungi and oomycete and has the potential for controlling grey mould rot of tomato plants and leaf scorch of pepper and could be useful in integrated control against diverse plant pathogenic fungi and oomycete. Significance and Impact of the Study: This study showed the potential that X. bovienii YL002 can be used to control the grey mould rot caused by B. cinerea on tomato plants and leaf scorch caused by P. capsici on pepper plants with the objective to reduce treatments with chemical fungicides.     

Purification of p47f,a Necrosis‐Inducing Protein Fraction from the Secretome of Phytophthora capsici

The oomycete Phytophthora capsici causes wilting disease in chilli pepper and another solanaceous plants, with important economic consequences. Although much investigation has been conducted about this pathogen, little is still known about which of its proteins are involved in the infection process. In this study, the bioassay‐guided fractionation of the secretome of P. capsici resulted in the purification of a phytotoxic protein fraction designated as p47f, capable of inducing wilting and necrosis on leaves of Capsicum chinense Jacq, and having a 47 kDa polypeptide with proteolytic activity as the major component. The isolated p47f fraction induced DNA degradation and decreased cell survival of C. chinense cell suspension culture. Sequencing of p47f indicated the presence of 15 proteins, which could be grouped into seven classes including a protease group, cell wall remodelling proteins and the transglutaminase elicitor M81D, among others. This is the first report of P. capsici secreting proteins that modulate cell responses mediated by ROS in the host.     

Isolation and Identification of Bacillus amyloliquefaciens IBFCBF‐1 with Potential for Biological Control of Phytophthora Blight and Growth Promotion of Pepper

In this study, 76 bacterial strains were isolated from the rhizosphere soil of pepper. Of these, 23 bacterial isolates capable of inhibiting Phytophthora capsici growth were selected. Among the antagonistic bacteria, one strain, IBFCBF‐1 showed the strongest antagonistic activity, and was identified as Bacillus amyloliquefaciens based on the results of 16S rRNA gene sequence analysis, physiological and biochemical testing, and morphological characteristics. When tested with a dual‐culture method and with laboratory greenhouse studies, the strain IBFCBF‐1 was found to be a potential biocontrol agent for controlling the plant pathogen, P. capsici. Moreover, it showed high efficiency and broad‐spectrum antifungal properties in vitro. Under greenhouse conditions, IBFCBF‐1 could significantly promote the growth of pepper seedlings, and was able to solubilize phosphate, and produce indole acetic acid (IAA) and ammonia. This study clearly demonstrated that IBFCBF‐1 is a potential candidate exhibiting phytophthora blight‐suppressive and plant growth‐promoting effects on pepper.     

Control of Late Blight (Phytophthora capsici ) in Pepper Plant with a Compost Containing Multitude of Chitinase-producing Bacteria

Compost sustaining a multitude of chitinase-producing bacteria was evaluated in a greenhouse study as a soil amendment for the control of late blight (Phytophthora capsici L.) in pepper (Capsicum annuum L.). Microbial population and exogenous enzyme activity were measured in the rhizosphere and correlated to the growth and health of pepper plant. Rice straw was composted with and without a chitin source, after having been inoculated with an aliquot of coastal area soil containing a known titer of chitinase-producing bacteria. P. capsici inoculated plants cultivated in chitin compost-amended soil exhibited significantly higher root and shoot weights and lower root mortality than plants grown in pathogen-inoculated control compost. Chitinase and β-1,3-glucanase activities in rhizosphere of plants grown in chitin compost-amended soil were twice that seen in soil amended with control compost. Colony forming units of chitinase-producing bacteria isolated from rhizosphere of plants grown in chitin compost-amended soil were 10³ times as prevalent as bacteria in control compost. These results indicate that increasing the population of chitinase-producing bacteria and soil enzyme activities in rhizosphere by compost amendment could alleviate pathogenic effects of P. capsici.     

Pre-plant bacterisation: a strategy for delivery of beneficial endophytic bacteria and production of disease-free plantlets of black pepper (Pipernigrum L.)

Naturally occurring endophytic bacteria from black pepper vines were found to exhibit strong antagonistic activities against Phytophthora capsici and Radopholus similis. In order to deliver these bacterial strains, as well as to produce disease-free plantlets of black pepper, a pre-plant stem and root bacterisation was standardised. Stem bacterisation with endophytic Pseudomonas spp. was found to suppress P. capsici infection (over 90% reduction in lesion length) on cut shoots. Pre-plant root bacterisation with Pseudomonas aeruginosa, Pseudomonas putida and Bacillus megaterium yielded over 60% of plantlets free from P. capsici infection on roots. Curtobacterium luteum and B. megaterium recorded over 70% reduction of nematode population in soil with concomitant production of over 65% of nematode-free plantlets. Besides protecting the plants from the pathogens, the bacteria were also found to enhance the growth of rooted cuttings. The biocontrol potential of the above endophytic bacteria and their exploitation for disease management in the black pepper nursery are discussed.     

Inoculation of Paenibacillus illinoisensis alleviates root mortality, activates of lignification-related enzymes, and induction of the isozymes in pepper plants infected by Phytophthora capsici

To investigate the variations of the enzymes responsible for lignification, after inoculation with Phytophthora capsici and/or Paenibacillus illinoisensis KJA-424, in relation to biocontrol of Phytophthora blight in pepper, roots of two-month-old plants were inoculated with P. capsici inoculation (P), and co-inoculation of P. capsici and P. illinoisensis cell cultures (P + A). Root mortality of pepper plants induced by inoculation of P. capsici was completely recovered by co-inoculation with antagonistic KJA-424. At day 7, peroxidase (POD) activity increased by 36.7% in P-treated roots but by 7.1% only in P + A-treated, compared with control. Polyphenol oxidase (PPO) activity increased for 3 days and then drastically decreased in P-treated roots but maintained a constant level in control and P + A-treated. At day 7, PPO activity in P-treated leaves decreased but recovered to the level of control in the P + A-treated. Three major POD isozymes (45, 53, and 114 kDa) were shown in P-treated roots, while two major (53 and 114 kDa) in control and P + A-treated, suggesting that the 45 kDa of POD was actively induced in P-treated roots but not induced in P + A-treated roots. A PPO isozyme of 80 kDa was induced in P-treated roots but not induced by co-treated with KJA-424. In leaves, the POD isozyme of 45 kDa appears to be systemically induced in P-treated only. The PPO isozyme of 80 kDa in leaves was not induced by pathogen challenge but recovered by co-inoculated with P. illinoisensis. All these results suggest that the inoculation of an antagonist, P. illinoisensis alleviates root mortality, activates of lignification-related enzymes and induction of the isozymes in pepper plants infected by P. capsici.     

Taxonomic Characterization and Experimental Host Ranges of Four Newly Recorded Species of Alternaria from Japan

Alternaria fungi are important plant pathogens. Here, we identified three species new to the Japanese mycoflora: Alternaria celosiae, Alternaria crassa and Alternaria petroselini. We proposed a new name for A. celosiae (E.G. Simmons & Holcomb) Lawrence, Park & Pryor, a later homonym of A. celosiae (Tassi) O. S?vul. To characterize these and a fourth morphological taxon, Alternaria alstroemeriae, which was recently added to Japan's mycoflora, an integrated species concept was tested. We determined the host range of each isolate using inoculation tests and analysed its phylogenetic position using sequences of the internal transcribed spacer rDNA. The pathogenicity of our A. alstroemeriae isolate was strictly limited to Alstroemeria sp. (Alstroemeriaceae), but the species was phylogenetically indistinguishable from other small‐spored Alternaria. Alternaria celosiae on Celosia argentea var. plumosa (Amaranthaceae) was also pathogenic to Amaranthus tricolor, to Alternanthera paronychioides and weakly to Gomphrena globosa (all Amaranthaceae) and formed a clade with the former Nimbya celosiae. Alternaria crassa on Datura stramonium (Solanaceae) was also pathogenic to Brugmansia × candida and Capsicum annuum in Solanaceae, but not to other confamilial plants; phylogenetically it belonged to a clade of large‐spored species with filamentous beaks. Morphological similarity, phylogenetic relationship and experimental host range suggested that A. crassa, Alternaria capsici and Alternaria daturicola were conspecific. Alternaria petroselini on Petroselinum crispum (Apiaceae) was pathogenic to five species in the tribe Apieae as well as representatives of Bupleureae, Coriandreae, Seliaeae and Scandiceae in Apiaceae. Both phylogeny and morphology suggested conspecificity between A. petroselini and Alternaria selini.     

Diversity and activity of biosurfactant-producing Pseudomonas in the rhizosphere of black pepper in Vietnam

Aims: Phytophthora capsici is a major pathogen of black pepper and zoospores play an important role in the infection process. Fluorescent pseudomonads that produce biosurfactants with zoosporicidal activities were isolated from the black pepper rhizosphere in Vietnam, and their genotypic diversity and potential to control Phy. capsici root rot was determined. Methods: Biosurfactant‐producing pseudomonads were genotypically and biochemically characterized by BOX‐polymerase chain reaction (PCR), 16S‐rDNA sequencing, reverse‐phase‐high‐performance liquid chromatography and liquid chromatography‐masss spectrometry analyses. Results: Biosurfactant‐producing fluorescent pseudomonads make up c. 1.3% of the culturable Pseudomonas population in the rhizosphere of black pepper. Although BOX‐PCR revealed substantial genotypic diversity, the isolates were shown to produce the same biosurfactants and were all identified as Pseudomonas putida. When applied to black pepper stem cuttings, several of the biosurfactant‐producing strains provided significant disease control. In absence of the disease, several of the bacterial strains promoted shoot and root growth of black pepper stem cuttings. Conclusions: Biosurfactant‐producing pseudomonads indigenous to the rhizosphere of black pepper plants are genotypically diverse and provide a novel resource for the control of Phy. capsici root rot and growth promotion of black pepper stem cuttings. Significance and Impact of the Study: The results of this study provide a strong basis for further development of supplementary strategies with antagonistic bacteria to control foot and root rot of black pepper and to promote plant growth.     

Phytophthora Crown and Root Rot of Apricot Caused by Phytophthora palmivora in Turkey

Forty‐nine Phytophthora isolates were obtained from roots and crown of apricot trees with symptoms of decline grown in commercial orchards in Malatya, Elaz?? and Diyarbak?r provinces, Turkey, in 2011 and 2013. All of the recovered isolates were identified as Phytophthora palmivora on the basis of morphological characteristics. Blast analysis of ITS region sequences of rDNA of 5 isolates revealed 100% identity with a reference isolates of P. palmivora from GenBank. Isolates of P. palmivora were pathogenic on 12‐month‐old wild apricot rootstock ‘Zerdali’ plants that were wound inoculated on the roots and on the crown. This study demonstrated that P. palmivora is the cause of the crown and root rot found on apricot in Turkey. To our knowledge, this is the first report of P. palmivora on this host plant.     

Grafting on CM‐334 controls serrano chili wilting caused by Phytophthora capsici and changes phenology but does not affect fruit yield

Grafting is an alternative method of plant propagation used to prevent soil‐borne diseases. This technique can improve the development of plants and therefore improve fruit yield and quality; however, several studies report that when a plant is grafted, there may be compatibility problems and changes in the phenological pattern of the crop and fruit yield and quality with respect to non‐grafted plants. There are no reports in the literature on the behaviour of serrano chilli grafted on varieties resistant to Phytophthora capsici. In this study, we evaluated the phenological behaviour and response to inoculation with P. capsici in commercial serrano chilli (Camino Real, Harris Moran) grafted or not on CM‐334 as a strategy to explore the possibility of incorporating the use of grafts in the production systems of serrano chilli. The plants were grafted at 55–60 days and maintained for 13 days in a curing chamber for the acclimatization process. At 36 and 43 days after transplantation, they were inoculated with the pathogen (300,000 zoospores/plant). None of plants grafted and inoculated with the pathogen showed wilt symptoms. All plants not grafted and inoculated with P. capsici died. There was a significant reduction in the production of leaves and flowers in the grafted plants, in relation to the non‐inoculated and non‐grafted plants, as well as a temporary delay in the beginning of fruit production with respect to the non‐grafted plants, but this delay did not affect the overall yield of the crop.     

Acibenzolar-S-methyl induced resistance to <Emphasis Type="Italic">Phytophthora capsici</Emphasis> in pepper leaves

The leaves of pepper (Capsicum anuum L.) were inoculated with Phytophthora capsici Leonian 3 d after treatment with acibenzolar-S-methylbenzo [1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester (ASM) and resistance to Phytophthora blight disease was investigated. Results showed that P. capsici was significantly inhibited by ASM treatment by up to 45 % in planta. The pepper plants responded to ASM treatments by rapid and transient induction of L-phenylalanine ammonia-lyase (PAL), increase in total phenol content and activities of chitinase and β-1,3-glucanase. No significant increases in enzyme activities were observed in water-treated control plants compared with the ASM-treated plants. Therefore it may be suggested that ASM induces defense-related enzymes, PAL activity, PR proteins and phenol accumulation in ASM-treated plants and contribute to enhance resistance against P. capsici.     

Occurrence of Phytophthora Root and Stem Rot of Kiwifruit in Turkey

Vine decline of kiwifruit was observed in an orchard in Bart?n province of Turkey. Affected vines exhibited poor terminal growth, leaf discoloration and various degrees of dieback, including complete vine death. Symptoms were observed in the field on roots, crowns and stems. Two Phytophthora species were isolated from decayed cortical roots and lower stems of kiwifruits. They were identified as Phytophthora cryptogea and Phytophthora megasperma by their morphological characteristics and the analysis of sequences of the internal transcribed spacer (ITS) region of the rDNA. Pathogenicity of the isolates was tested by stem inoculation on kiwifruit seedlings. After 4 weeks, cankers developed in the plants inoculated with P. cryptogea, while no cankers formed in those inoculated with P. megasperma and in control plants. This is the first report of P. cryptogea causing root and stem rot of kiwifruit in Turkey.     

Bulb and Root Rot in Lily (Lilium longiflorum) and Onion (Allium cepa) in Israel

In the past 10 years, there has been a substantial increase in reports, from growers and extension personnel, on bulb and root rots in lily (Lilium longiflorum) in Israel. Rot in these plants, when grown as cut flowers, caused serious economic damage expressed in reduction in yield and quality. In lily, the fungal pathogens involved in the rot were characterized as binucleate Rhizoctonia AG‐A, Rhizoctonia solani, Pythium oligandrum, Fusarium proliferatum (white and purple isolates) and F. oxysporum, using morphological and molecular criteria. These fungi were the prevalent pathogens in diseased plants collected from commercial greenhouses. Pathogenicity trials were conducted on lily bulbs and onion seedlings under controlled conditions in a greenhouse to complete Koch's postulates. Disease symptoms on lily were most severe in treatments inoculated with binucleate Rhizoctonia AG‐A, P. oligandrum and F. proliferatum. Plant height was lower in the above treatments compared with the control plants. The least aggressive fungus was R. solani. In artificial inoculations of onion, seedling survival was significantly affected by all fungi. The most pathogenic fungus was F. proliferatum w and the least were isolates of F. oxysporum (II and III). All fungi were successfully re‐isolated from the inoculated plants.     

Functional analysis of Pcipg2 from the straminopilous plant pathogen Phytophthora capsici

Phytophthora capsici causes serious diseases in numerous crop plants. Polygalacturonases (PGs) are cell wall‐degrading enzymes that play an important role in pathogenesis in straminopilous pathogens. To understand PGs as they relate to the virulence of P. capsici, Pcipg2 was identified from a genomic library of a highly virulent P. capsici strain. Pcipg2 was strongly expressed during symptom development after the inoculation of pepper leaves with P. capsici. The wild protein (PCIPGII) was obtained from the expression of pcipg2 and found that increasing activity of PGs in PCIPGII‐treated pepper leaves was consistent with increasing symptom development. Asp residues in active sites within pcipg2 affected PCIPGII activity or its virulence on pepper leaves. Results show that pcipg2 is an important gene among pcipg genes, and illustrate the benefit of analyzing mechanisms of pathogenicity during the period of host/parasite interaction. genesis 47:535–544, 2009. © 2009 Wiley‐Liss, Inc.     

Changes in pathogenesis-related proteins in pepper plants with regard to biological control of phytophthora blight with <Emphasis Type="Italic">Paenibacillus illinoisensis</Emphasis>

To evaluate the biocontrol effectiveness of chitinase-producing bacterium, Paenibacillus illinoisensis strain KJA-424 against pathogenic strain of Phytophthora capsici in pepper plants, growth response and kinetics of pathogen related (PR) proteins were estimated after inoculation with P. capsici (P), and with a combination of P. capsici and strain KJA-424 cell culture (P+A). Fresh weight and chlorophyll content in shoots at P+A-treated plants significantly increased by 23.4 and 34.2%, respectively after 7days of inoculation, compared to P-treated plants. Root mortality in P+A-treated plants was significantly reduced compared to P-treated plants. Seven days after inoculation, the activities of -1,3-glucanase, cellulase and chitinase in P-treated roots had decreased by 54.8, 36.5 and 52.8%, respectively, compared to P+A-treated roots, while those in P-treated leaves increased by 22.8, 36.3 and 23.8%, respectively, compared to those in P+A-treated leaves. The activities of -1,3-glucanase, cellulase and chitinase in roots are negatively correlated with root mortality. All these results suggest that the inoculation of an antagonist, P. illinoisensis alleviates root mortality, reduction of PR proteins in roots, and activates of PR proteins in leaves infected by P. capsici.