Soilborne Phytopathogenic Fungi Research Articles

Exquisite binding specificity of Sclerotium rolfsii lectin toward TF-related O-linked mucin-type glycans

Sclerotium rolfsii lectin (SRL), a secretory protein from the soil borne phytopathogenic fungus Sclerotium rolfsii, has shown in our previous studies to bind strongly to the oncofetal Thomson-Friedenreich carbohydrate (Galβ1-3GalNAc-ser/thr, T or TF) antigen. TF antigen is widely expressed in many types of human cancers and the strong binding of SRL toward such a cancer-associated carbohydrate structure led us to characterize the carbohydrate binding specificity of SRL. Glycan array analysis, which included 285 glycans, shows exclusive binding of SRL to the O-linked mucin type but not N-linked glycans and amongst the mucin type O-glycans, lectin recognizes only mucin core 1, core 2 and weakly core 8 but not to other mucin core structures. It binds with high specificity to "α-anomers" but not the "β-anomers" of the TF structure. The axial C4-OH group of GalNAc and C2-OH group of Gal is both essential for SRL interaction with TF disaccharide, and substitution on C3 of galactose by sulfate or sialic acid or N-acetylglucosamine, significantly enhances the avidity of the lectin. SRL differs in its binding to TF structures compared to other known TF-binding lectins such as the Arachis hypogea (peanut) agglutinin, Agaricus bisporus (mushroom) lectin, Jackfruit, Artocarpus integrifolia (jacalin) and Amaranthus caudatus (Amaranthin) lectin. Thus, SRL has unique carbohydrate-binding specificity toward TF-related O-linked carbohydrate structures. Such a binding specificity will make this lectin a very useful tool in future structural as well as functional analysis of the cellular glycans in cancer studies.

In vitro Characterization of Trichoderma viride for Abiotic Stress Tolerance and Field Evaluation against Root Rot Disease in Vigna mungo L. Leo Daniel

Soil-borne phytopathogenic fungi pose serious threats to yield of several crops. Biological control is an ecofriendly approach in the effective management of crop diseases. Trichoderma viride is an important soil-borne fungus, which play an important role in antagonism by secretion of different hydrolytic enzymes. Black gram is an important pulse crop world-wide and its yield is severely affected by Macrophomina root rot. Abiotic stresses greatly influence the performance of biocontrol agents. T. viride was evaluated for its In vitro abiotic stress tolerance ability and its field bioefficacy against root rot disease in blackgram. Growth of T. viride decreased with increasing in salinity, temperature and drought. T. viride effectively inhibited the growth of R. solani (45%) and M. phaseolina (40%) under In vitro conditions. T. viride was compatible with 0.25% mancozeb, 1.0% copper oxy chloride and metalaxyl. Among three doses, plants treated with 6 g.kg-1 of T. viride showed highest yield of 1375 kg.ha-1 and lowest root rot incidence of 14.77% which were statistically on par with 4 g.kg-1 T. viride treated plants. To conclude, this study identified an abiotic stress tolerant T. viride for effective management of root rot disease and enhanced yield of Vigna mungo when applied as seed dresser at a concentration of 4g kg-1 under field conditions.

Diversity of Culturable Chitinolytic Bacteria from Rhizospheres of Agronomic Plants in Japan

A total of 100 isolates of chitinolytic bacteria were obtained from the rhizospheres of various agronomic plants, and the 16S rRNA gene sequences of these isolates were determined. Phylogenetic analyses revealed that 81 isolates belonged to the classes Betaproteobacteria (39 isolates) and Gammaproteobacteria (42 isolates). Of the remaining 19 isolates, 16 belonged to the phylum Firmicutes. Clustering analysis identified 6 and 3 operational taxonomic units (OTUs) in Gammaproteobacteria and Betaproteobacteria, respectively, at the genus level. The majority of chitinolytic bacteria in Gammaproteobacteria belonged to the genera Serratia, Stenotrophomonas, and Lysobacter (14, 15, and 7 isolates, respectively) while those in Betaproteobacteria belonged to the genus Mitsuaria (37 isolates). The 16 isolates placed in Firmicutes belonged to 2 genera, Paenibacillus and Bacillus (8 isolates each). The isolates in the remaining OTUs belonged to the genera Erwinia, Aeromonas, Pseudomonas, Achromobacter, Flavobacterium, and Microbacterium, in less abundance. These results showed a wide distribution of culturable chitinolytic bacteria in the rhizospheres of various agronomic plants. Considering the potential antagonistic activity of chitinolytic enzymes against phytopathogenic fungi, which is exhibited by fungal cell wall degradation, the above-mentioned native chitinolytic bacteria in rhizospheres could potentially be utilized for the biological control of soil-borne phytopathogenic fungi.

Transmission electron microscopic study of the cytological changes in Sclerotium rolfsii parasitized by a biocontrol fungus Trichoderma sp

There is a recent trend towards the use of eco-friendly biological control agents for protecting crops from pest and disease, especially soil-borne plant pathogens, as an alternative to existing chemical methods. Among various biocontrol agents, Trichoderma, which has multiple mechanisms for the biocontrol of phytopathogens, is used widely. The present study explores the role of Trichoderma sp. in inducing cytological changes in fungal plant pathogens during parasitization. In dual culture plates, all the fungal isolates (SE6, KT6, KT28, and BRT11) along with a standard culture of T. harzianum were able to antagonize and mycoparasitize two soil-borne fungal phytopathogens (Sclerotium rolfsii and Rhizoctonia solani) of chickpea wilt complex. The suppression of S. rolfsii was slower than that of R. solani. The interaction between T. harzianum and sclerotia of S. rolfsii was studied by light microscopy and transmission electron microscopy (TEM). Ultra-structural examinations revealed that growth and development of Trichoderma resulted in extensive host cell alterations, such as retraction, aggregation and disintegration of cytoplasmic contents. Lysis and deformation of hyphal cell wall, degradation and disappearance of cytoplasmic contents and loss of cellular integrity in sclerotia of S. rolfsii paratisized by T. harzianum is clearly apparent from transmission electron micrographs.

Screening for candidate bacterial biocontrol agents against soilborne fungal plant pathogens

Over the years, many bacterial isolates have been evaluated as potential biocontrol agents against soilborne fungal phytopathogens. However, few of them were ultimately successful after evaluation in field trials. One of the major reasons for this failure is the lack of appropriate screening procedures to select the most suitable microorganisms for disease control in diverse soil environments. For this reason, the study of bacterial screening has a future that is characterised by many technical and conceptual challenges. In this review, we summarise and discuss the convenience of use of the main screening methods currently applied to select bacterial candidates for biocontrol of fungal and oomycete soilborne phytopathogens. Also, a comparative case study of the application of different screening methods applied to an experimental pathosystem is shown, revealing the success of bacterial candidates selected by different strategies for biocontrol of the phytopathogenic fungus Rosellinia necatrix in avocado plants. Screening for antagonism against this fungal pathogen, one of the more straightforward methods used for the selection of bacterial biocontrol agents, was proven to be a valid strategy for this experimental system.

Purification and Characterization of Chitinase from Paenibacillus sp. D1

A 56.56-kDa extracellular chitinase from Paenibacillus sp. D1 was purified to 52.3-fold by ion exchange chromatography using SP Sepharose. Maximum enzyme activity was recorded at pH5.0 and 50°C. MALDI-LC-MS/MS analysis identified the purified enzyme as chitinase with 60% similarity to chitinase Chi55 of Paenibacillus ehimensis. The activation energy (E (a)) for chitin hydrolysis and temperature quotient (Q (10)) at optimum temperature was found to be 19.14kJ/mol and 1.25, respectively. Determination of kinetic constants k (m), V (max), k (cat), and k (cat)/k (m) and thermodynamic parameters ΔH*, ΔS*, ΔG*, ΔG*(E-S), and ΔG*(E-T) revealed high affinity of the enzyme for chitin. The enzyme exhibited higher stability in presence of commonly used protectant fungicides Captan, Carbendazim, and Mancozeb compared to control as reflected from the t (1/2) values suggesting its applicability in integrated pest management for control of soil-borne fungal phytopathogens. The order of stability of chitinase in presence of fungicides at 80°C as revealed from t (1/2) values and thermodynamic parameters E (a(d)) (activation energy for irreversible deactivation), ΔH*, ΔG*, and ΔS* was: Captan > Carbendazim > Mancozeb > control. The present study is the first report on thermodynamic and kinetic characterization of chitinase from Paenibacillus sp. D1.

Inhibitory effects of potassium silicate on five soil-borne phytopathogenic fungi in vitro

Silicon (Si) can reduce the severity of fungal diseases in field crops, but the mechanism by which Si protects plants from fungal diseases is not well understood. This study investigated the effect of potassium silicate on the growth of five soilborne phytopathogenic fungi in vitro. Results showed that the growth of four of the fungal isolates (Rhizoctonia solani, Pestalotiopsis clavispora, Fusarium oxysporum and Fusarium oxysporum f. sp. fragariae) was signficantly (P < 0.05) inhibited on potassium silicate amended PDA plates. However, when the pH of potassium silicate amended PDA was reduced so that it was equivalent to unamended PDA (control), there was no difference in fungal growth between the two treatments. This indicated that the inhibition of fungal growth on potassium silicate amended PDA was due to a pH effect. Our results imply that reductions in fungal disease after field plants are treated with low concentrations of Si are probably not due to fungistatic effects of Si, but rather due to other mechanisms such as Si acting as a physical barrier against pathogen penetration or Si-induced defense response in plants.

Mycoparasitism of Endophytic Fungi Isolated From Reed on Soilborne Phytopathogenic Fungi and Production of Cell Wall-Degrading Enzymes In Vitro

Antagonism of three endophytic fungi isolated from common reed (Phragmites australis) against eight soilborne pathogenic fungi was investigated on potato dextrose agar by light microscopy, scanning electron microscopy, and transmission electron microscopy. Inhibitory zones were not observed. The microscopical studies suggested that the endophytes inhibit growth of soilborne pathogens by means of coiling around hyphae and, after penetration, the degradation of hyphal cytoplasm. Since penetration of hyphae seems to play a major role in parasitism, we studied the production of cell wall degrading enzymes by the three endophytes. Choiromyces aboriginum produced higher activities of beta-1,3-glucanases compared to Stachybotrys elegans and Cylindrocarpon sp. For C. aboriginum and S. elegans, colloidal chitin was the best substrate for the induction of beta-1,3-glucanases and chitinases, respectively. This result suggests that mycoparasitism by endophytes on soilborne plant pathogens can be explained by their mycoparasitic activity.

Sobrevivência de fungos fitopatogênicos habitantes do solo, em microcosmo, simulando solarização com prévia incorporação de materiais orgânicos

Os fungos fitopatogênicos habitantes do solo podem sobreviver por vários anos nesse ambiente por meio de estruturas de resistência, causando perdas em muitas culturas, por vezes, inviabilizando o pleno aproveitamento de vastas áreas agrícolas. O uso de materiais orgânicos no solo consorciado com a técnica de solarização propicia a retenção de compostos voláteis fungitóxicos emanados da rápida degradação dos materiais e que são letais a vários fitopatógenos. O objetivo deste experimento foi à prospecção de novos materiais orgânicos que produzissem voláteis fungitóxicos capazes de controlar fungos fitopatogênicos habitantes do solo, em condições de associação com a simulação da técnica de solarização (microcosmo). Portanto, o presente trabalho consistiu de seis tratamentos (Solarizado; Solarizado+Brócolos; Solarizado+Eucalipto; Solarizado+Mamona; Solarizado+Mandioca e Laboratório) e cinco períodos (0, 7, 14, 21 e 28 dias) para avaliar a sobrevivência de quatro fungos de solo (Fusarium oxysporum f. sp. lycopersici Raça 2; Macrophomina phaseolina; Rhizoctonia solani AG-4 HGI e Sclerotium rolfsii). Em cada uma das duas câmaras de vidro (microcosmo) por dia avaliado continha uma bolsa de náilon contendo as estruturas de resistência de cada fitopatógeno. Estruturas dos fitopatógenos foram mantidas também em condições de laboratório como referencial de controle. Todos os materiais quando associados à simulação da solarização propiciaram o controle de todos os fitopatógenos estudados, entretanto, foi observado variação no controle dos fungos. O tratamento que apenas simulou a solarização não controlou nenhum fitopatógeno.

Evaluation of antifungal activity of food additives against soilborne phytopathogenic fungi

The efficacy of eight food additives as possible alternatives to synthetic fungicides for the control of soilborne pathogens, Fusarium oxysporum f. sp. melonis, Macrophomina phaseolina, Rhizoctonia solani, and Sclerotinia sclerotiorum was evaluated in this study. A preliminary selection of food additives was performed through in vitro tests. The ED50, minimum inhibition concentration (MIC), and minimum fungicidal concentration (MFC) values showed that ammonium bicarbonate and potassium sorbate were more toxic to soilborne pathogens compared to other food additives with few exceptions and, therefore selected for further testing in soil. The inhibitory and fungistatic efficacy potassium sorbate were higher than that of ammonium bicarbonate in in vitro tests. Potassium sorbate completely inhibited F. oxysporum f. sp. melonis, M. phaseolina, and R. solani at 0.6% in soil tests. In contrast ammonium bicarbonate at 0.6% was inferior compared to potassium sorbate. Ammonium bicarbonate achieved to control all fungi at 2% that is the highest concentration used in this study. Potassium sorbate showed higher toxicity to all fungi compared to ammonium bicarbonate in soil tests. Both ammonium bicarbonate and potassium sorbate increased the pH of soil. The rate of pH increase was higher in ammonium bicarbonate.

Biological potantial of some Iranian Trichoderma isolates in the control of soil borne plant pathogenic fungi

In this study the in vitro potential of six selected Iranian isolates of three species of Trichoderma (Trichoderma hamatum T614, T. hamatum T612, Trichoderma harzianum T447, T. harzianum T969, Trichoderma virens T523 and Trichoderma sp. T) were evaluated against five isolates of soil borne phytopathogenic fungi (Fusarium graminearum, Rhizoctonia solani (AG4 and AG5), Macrophomina phaseoli and Phytophtora cacturum) in dual culture techniques and through production of volatile and non-volatile inhibitors, and the pH and temperature effects on Trichoderma mycelial growth were also evaluated. All Trichoderma isolates had a marked statistical inhibitory effect on mycelial growth of the pathogens in dual culture compared with controls. Maximum inhibitions occurred in F. graminearum-T. hamatum T614 interaction. Significant pathogen colony growth inhibitions were observed when exposed to the trapped atmosphere from culture of the Trichoderma. F. graminearum was most susceptible to the volatile inhibitors produced by T. hamatum T612 (%inhibition = 48.65). Medium filtrate obtained the Trichoderma isolate culture also were effected on the pathogen species significantly. Maximum growth inhibition was observed in radial growth of F. graminearum by T. hamatum T612 non volatile metabolites (%inhibition = 38.3). Evaluation of pH and temperature effects on Trichoderma isolates mycelial growth showed that Trichoderma strains were found to be able to display activities under a wider range of pH values. Also, Trichoderma strains are mesophilic.

PltR expression modulated by the global regulators GacA, RsmA, LasI and RhlI in Pseudomonas sp. M18

Pseudomonas sp. M18 can suppress certain soil-borne phytopathogenic fungi by producing two antibiotics, pyoluteorin (Plt) and phenazine-1-carboxylic acid (PCA). pltR encodes an LysR-type transcriptional activator required for expression of divergently transcribed Plt biosynthetic genes. Here we provide evidence that Plt biosynthesis is negatively regulated by the quorum-sensing (QS) signal molecule (N-acyl homoserine lactone, AHL) synthase gene lasI and positively regulated by the orphan LuxR-type regulatory gene vqsR. pltR expression is modulated by four important global regulators including the two-component response regulatory gene gacA, the RNA-binding repressor RsmA, and the QS signal molecule synthase genes lasI and rhlI. pltR translation was almost completely inhibited in the gacA mutant M18G when compared with that in the wild-type strain of M18. We also observed significant enhancement of pltR translation in the rsmA mutant compared with wild-type strain M18. Moreover, mutation in lasI or rhlI gave rise to a significantly elevated level of pltR transcription in strain M18. However, no obvious difference was observed in pltR expression between the vqsR mutant and the wild-type of strain M18. These results highlight the fact that PltR likely at least partly plays an important mediator role in Gac/Rsm and the Las/Rhl regulatory pathways involved in Plt biosynthesis.

Antifungal Effects of Volatile Compounds from Black Zira (Bunium persicum) and Other Spices and Herbs

The dish pack method, which measures growth inhibition or promotion effects of volatile compounds on germinating seeds, was applied to measure the antifungal effects of 52 dried samples of spices and herbs against a soil-borne phytopathogenic fungus, Fusarium oxysporum. Black zira showed the strongest effect, followed by cumin and cardamom. Headspace sampling and gas chromatography-mass spectrometry analysis of black zira identified seven volatile compounds, gamma-terpinene, limonene, p-cymene, beta-pinene, alpha-pinene, cuminaldehyde, and myrcene. Among these, cuminaldehyde and p-cymene showed the strongest antifungal activities against F. oxysporum, suggesting roles in the antifungal activity of black zira. The same compounds also showed antifungal activities against another soil-borne phytopathogenic fungus, Verticillium dahliae, and foliar phytopathogenic fungi, Botrytis cinerea and Alternaria mali. The total activity calculated from the concentration of cuminaldehyde contained in black zira and its EC(50) against F. oxysporum demonstrated that cuminaldehyde is the main antifungal compound detected in black zira.

Isolation and characterization of antagonistic Bacillus subtilis strains from the avocado rhizoplane displaying biocontrol activity

This study was undertaken to isolate Bacillus subtilis strains with biological activity against soil-borne phytopathogenic fungi from the avocado rhizoplane. A collection of 905 bacterial isolates obtained from the rhizoplane of healthy avocado trees, contains 277 gram-positive isolates. From these gram-positive isolates, four strains, PCL1605, PCL1608, PCL1610 and PCL1612, identified as B. subtilis, were selected on the basis of their antifungal activity against diverse soil-borne phytopathogenic fungi. Analysis of the antifungal compounds involved in their antagonistic activity showed that these strains produced hydrolytic enzymes such as glucanases or proteases and the antibiotic lipopeptides surfactin, fengycin, and/or iturin A. In biocontrol trials using the pathosystems tomato/Fusarium oxysporum f.sp. radicis-lycopersici and avocado/Rosellinia necatrix, two B. subtilis strains, PCL1608 and PCL1612, both producing iturin A, exhibited the highest biocontrol and colonization capabilities. Diverse antagonistic B. subtilis strains isolated from healthy avocado rhizoplanes have shown promising biocontrol abilities, which are closely linked with the production of antifungal lipopeptides and good colonization aptitudes. This is one of the few reports dealing with isolation and characterization of B. subtilis strains with biocontrol activity against the common soil-borne phytopathogenic fungi F. oxysporum f.sp. radicis-lycopersici and R. necatrix.

Produção e avaliação da sobrevivência de estruturas de resistência de fungos fitopatogênicos habitantes do solo

Os fungos fitopatogênicos habitantes do solo causam grandes perdas em culturas econômicas. Estes organismos produzem estruturas de resistência na ausência de hospedeiros e/ou nas condições climáticas desfavoráveis. Isto permite a sobrevivência destes organismos no solo por longos períodos de tempo. A presença das estruturas de resistência inviabiliza o controle do patógeno. O objetivo deste trabalho foi desenvolver metodologias para produzir e também para avaliar a sobrevivência das estruturas de resistência dos fungos Fusarium oxysporum f.sp. lycopersici raça 2, Macrophomina phaseolina, Rhizoctonia solani AG4 HGI, Sclerotium rolfsii, Sclerotinia sclerotiorum e Verticillium dahliae. O delineamento experimental foi inteiramente casualizado, constituído por 6 fungos, 8 épocas de avaliação (0, 15, 30, 60, 90, 120, 150 e 180 dias do início do experimento) e duas condições: campo e laboratório. No campo, a 10 cm de profundidade, para cada época de avaliação, foram enterradas duas bolsas contendo estruturas de cada fungo. No laboratório, foram mantidos dois frascos para cada patógeno. O parâmetro avaliado foi a sobrevivência das estruturas dos fungos. Constatou-se que todas as metodologias desenvolvidas refletiram de forma positiva quanto a sobrevivência dos fungos no campo e na avaliação dessa característica em condições de laboratório.

Volatiles of bacterial antagonists inhibit mycelial growth of the plant pathogen Rhizoctonia solani

Bacterial antagonists are bacteria that negatively affect the growth of other organisms. Many antagonists inhibit the growth of fungi by various mechanisms, e.g., secretion of lytic enzymes, siderophores and antibiotics. Such inhibition of fungal growth may indirectly support plant growth. Here, we demonstrate that small organic volatile compounds (VOCs) emitted from bacterial antagonists negatively influence the mycelial growth of the soil-borne phytopathogenic fungus Rhizoctonia solani Kühn. Strong inhibitions (99-80%) under the test conditions were observed with Stenotrophomonas maltophilia R3089, Serratia plymuthica HRO-C48, Stenotrophomonas rhizophila P69, Serratia odorifera 4Rx13, Pseudomonas trivialis 3Re2-7, S. plymuthica 3Re4-18 and Bacillus subtilis B2g. Pseudomonas fluorescens L13-6-12 and Burkholderia cepacia 1S18 achieved 30% growth reduction. The VOC profiles of these antagonists, obtained through headspace collection and analysis on GC-MS, show different compositions and complexities ranging from 1 to almost 30 compounds. Most volatiles are species-specific, but overlapping volatile patterns were found for Serratia spp. and Pseudomonas spp. Many of the bacterial VOCs could not be identified for lack of match with mass-spectra of volatiles in the databases.

First Report of Wilt and Stem Canker of Poinsettia (Euphorbia pulcherrima) Caused by Phytophthora nicotianae in Puerto Rico.

Poinsettias are grown extensively in greenhouses in Puerto Rico, a five million dollar industry during the winter season of 2005. More than 2,000 'Freedom' plants from an ornamental nursery near Aibonito exhibited severe wilt and dieback symptoms. The disease was found in 5 of 12 surveyed greenhouses where severity ranged from 50 to 100% and occurred regardless of the use of metalaxyl and mefenoxam. Symptoms during the growth phase included stunted plants, thin stems, chlorotic leaves, and brown roots. During flower bract development, symptoms consisted of leaf wilting often in sectors, stem canker, and purple-to-black lesions that extended from the stem to the petioles followed by soft rot. Isolations from symptomatic stem and root tissue were made on corn meal agar (CMA) (17 g/liter) amended with pimaricin (10 mg/liter), ampicillin (250 mg/liter), rifampicin (10 mg/liter), pentachloronitrobenzene (100 mg/liter), and hymexazol (50 mg/liter) (PARPH) (1). Pure cultures were obtained by hyphal-tip transfers onto CMA-PARPH. Colonies grown on acidified potato dextrose agar (APDA) at 25°C were arachnoid with abundant aerial mycelium and sporangia. Sporangia formed singly or in a loose sympodium on long sporangiophores and were papillate, mostly persistent, or caducous with short pedicel. The shape of sporangia varied from ellipsoidal to elongated ovoid, with a length/breath ratio of 16.5 × 14.2 μm. Large, spherical, terminal, and intercalary chlamydospores readily formed in APDA and PARPH. The isolate was identified as Phytophthora nicotianae on the basis of morphological and molecular characteristics. Molecular identification by sequence analysis of the internal transcribed spacer rDNA region confirmed the identity of the isolate P210.1 as P. nicotianae (GenBank Accession No. DQ485412). Pathogenicity tests were conducted with isolates P210.1 and IG grown on APDA for 7 days at 24°C. Isolates were then covered with sterile water for 48 h to induce sporangia and zoospore production at 24°C and continuous light. A suspension of 10 ml of approximately 120 sporangia ml-1 were added to pots containing 2.2 kg of a 1:1 mixture of field soil and soilless media (Promix) composed of peat moss vermiculite and perlite. One healthy poinsettia rooted stem and one 2-month-old plant were transplanted individually into the inoculated soil and pots were flooded with sterile water for 24 h. A noninoculated control was included, each isolate and control were replicated five times. First symptoms (7 to 10 days after inoculation) included brown, limited root growth and lower stem discoloration. Plants (30- to 45-day-old) show a decline in vigor and rapid death of branches that are similar symptoms to those observed on infected poinsettias in commercial greenhouses. P. nicotianae was reisolated from infected root, crown, and stem tissue on PARPH media. A strict sanitation program was recommended to prevent recontamination. To our knowledge, this is the first report of P. nicotianae on poinsettia in Puerto Rico. Reference: D. J. Mitchell and M. E. Kannwischer-Mitchel, Phytophthora. Page:31 in: Methods for Research on Soilborne Phytopathogenic Fungi. L. L. Singleton et al., eds. The American Phytopathological Society, St. Paul, MN, 1993.

Induction of Plant Defense Enzymes and Phenolics by Treatment With Plant Growth–Promoting Rhizobacteria Serratia marcescens NBRI1213

In greenhouse experiments, plant growth-promoting rhizobacteria (PGPR) Serratia marcescens NBRI1213 was evaluated for plant growth promotion and biologic control of foot and root rot of betelvine caused by Phytophthora nicotianae. Bacterization of betelvine (Piper betle L.) cuttings with S. marcescens NBRI1213 induced phenylalanine ammonia-lyase, peroxidase, and polyphenoloxidase activities in leaf and root. Qualitative and quantitative estimation of phenolic compounds was done through high-performance liquid chromatography (HPLC) in leaf and root of betelvine after treatment with S. marcescens NBRI1213 and infection by P. nicotianae. Major phenolics detected were gallic, protocatechuic, chlorogenic, caffeic, ferulic, and ellagic acids by comparison of their retention time with standards through HPLC. In all of the treated plants, synthesis of phenolic compounds was enhanced compared with control. Maximum accumulation of phenolics was increased in S. marcescens NBRI1213-treated plants infected with P. nicotianae. In a greenhouse test, bacterization using S. marcescens NBRI1213 decreased the number of diseased plants compared with nonbacterized controls. There were significant growth increases in shoot length, shoot dry weight, root length, and root dry weight, averaging 81%, 68%, 152%, and 290%, respectively, greater than untreated controls. This is the first report of PGPR-mediated induction of phenolics for biologic control and their probable role in protecting betelvine against P. nicotianae, an important soil-borne phytopathogenic fungus.

Biocontrol of Avocado Dematophora Root Rot by Antagonistic Pseudomonas fluorescens PCL1606 Correlates With the Production of 2-Hexyl 5-Propyl Resorcinol

A collection of 905 bacterial isolates from the rhizospheres of healthy avocado trees was obtained and screened for antagonistic activity against Dematophora necatrix, the cause of avocado Dematophora root rot (also called white root rot). A set of eight strains was selected on the basis of growth inhibitory activity against D. necatrix and several other important soilborne phytopathogenic fungi. After typing of these strains, they were classified as belonging to Pseudomonas chlororaphis, Pseudomonas fluorescens, and Pseudomonas putida. The eight antagonistic Pseudomonas spp. were analyzed for their secretion of hydrogen cyanide, hydrolytic enzymes, and antifungal metabolites. P. chlororaphis strains produced the antibiotic phenazine-1-carboxylic acid and phenazine-1-carboxamide. Upon testing the biocontrol ability of these strains in a newly developed avocado-D. necatrix test system and in a tomato-F oxysporum test system, it became apparent that P. fluorescens PCL1606 exhibited the highest biocontrol ability. The major antifungal activity produced by strain P. fluorescens PCL1606 did not correspond to any of the major classes of antifungal antibiotics produced by Pseudomonas biocontrol strains. This compound was purified and subsequently identified as 2-hexyl 5-propyl resorcinol (HPR). To study the role of HPR in biocontrol activity, two Tn5 mutants of P. fluorescens PCL1606 impaired in antagonistic activity were selected. These mutants were shown to impair HRP production and showed a decrease in biocontrol activity. As far as we know, this is the first report of a Pseudomonas biocontrol strain that produces HPR in which the production of this compound correlates with its biocontrol activity.

Crystallization and preliminary X-ray crystallographic analysis of Sclerotium rolfsii lectin.

Sclerotium rolfsii lectin (SRL), from the soil-borne phytopathogenic fungus S. rolfsii, has been crystallized. SRL crystals were grown by the hanging-drop vapour-diffusion method using an MPD-ammonium acetate mixture in Tris-HCl buffer pH 8.5. A complete data set from a single crystal at 100 K was collected to 1.1 A resolution using synchrotron radiation. Preliminary crystallographic analysis showed that the crystals belong to the tetragonal space group P4(2)2(1)2, with unit-cell parameters a = b = 99.81, c = 63.99 A and two molecules per asymmetric unit.