Inhibición del crecimiento y modificación genética de Phytophthora capsici usando quitosano de bajo grado de polimerización / Growth inhibition and genetic modification of Phytophthora capsici using chitosan with low degree of polymerization

Phytophtora capsici es un patógeno que incide sobre cultivos de la familia de las solanáceas causando pérdidas económicas en cultivos de pimientos, tomates, berenjenas y cur-cubitáceas. En este trabajo evaluamos el efecto del quitosano de bajo grado de polimerización (QBP) sobre el crecimiento de P. capsici y sobre la regulación génica de este fitopatógeno a nivel transcripcional. A una concentración de 0,4mg/l de QBP se obtuvo un 88% de inhibición en el crecimiento; concentraciones superiores a 1,6 mg/l inhibieron el crecimiento en un 100%. Mediante ensayos de cambio en la movilidad electroforética de ácidos nucleicos se comprobó que el quitosano interactúa con el ADN y el ARN del hongo frente a concentraciones entre 2 y 4 mg/l de ADN y entre 0,5 y 3 mg/l de ARN. Además, se efectuó un análisis de despliegue diferencial de los productos de amplificación por RT-PCR de los ARN mensajeros de P. capsici obtenidos en presencia o ausencia de QBP; este mostró cambios en el perfil de expresión inducidos por el tratamiento con quitosano. El análisis bioinformático de las secuencias de los transcritos expresados diferencialmente sugiere que el QBP afectó la regulación génica de elementos involucrados en la síntesis de quitina y de proteínas de unión a hidratos de carbono.

Phytophthora blight of peppers, caused by oomycete Phytophthora capsici, currently causes economic losses in crops such as peppers, tomatoes, eggplant and cucurbits. In this work, we evaluated the effect of chitosan with low degree of polymerization (LDP) on growth and gene expression of P. capsici cultures. LDP chitosan inhibited 88% of P. capsici mycelial growth at concentrations up to 0,4 mg/l, whereas at concentrations higher than 1,6 mg/l it completely inhibit growth. Gel mobility shift assays demonstrated that chitosan interacts with DNA and RNA of the fungus at concentrations ranging from 2 to 4 mg/l for DNA and 0,5 to 3 mg/l for RNA. The differential display analysis of RT-PCR-amplification products of P. capsici messenger RNA revealed changes in gene expression profiles after the chitosan treatment. Bioinformatic analysis of sequences from selected differentially-expressed bands showed the gene regulation of elements involved in chitin synthesis and carbohydrate-binding proteins.

In Vitro Antagonism of Phytophthora capsici And Fusarium solani by Bacterial Isolates from Sarawak

Aims: Phytophthora capsici and Fusarium solani are common fungal pathogens causing severe diseases that lead to economic loss in pepper industry, especially in Sarawak. In response to the infections, chemical approach is more common; nevertheless, biological control is more favorable to control fungal pathogens. Biological control approach greatly reduces the problems associated with chemical applications and it restores balance of the natural environment. Here we present the ongoing work to study the action of antagonistic bacteria, Bacillus sp. and Pseudomonas sp., that produce volatile and non-volatile antifungal compounds against P. capsici and F. solani on pepper plants. Methodology and results: A total of seven bacterial candidates were isolated from different locations and tested for their antagonistic properties against P. capsici and F. solani in a dual culture assay and extracellular metabolite test. Extracellular hydrolytic enzymes production was also monitored and followed by genotypic indentification. Preliminary antagonism tests indicated that bacterial isolate Pep3 and Pep4 inhibit up to 50% of the growth of P. capsici and F. solani as compared to the control. Subsequent investigation on extracellular hydrolytic enzyme production revealed that both bacterial isolates are capable of secreting hydrolytic enzymes. Microscopic and genotypic analyses identified the bacterial isolates Pep3 as Bacillus amyloliquefaciens (KJ461444) and Pep4 as Pseudomonas pachastrellae (KM460937). Conclusion, significance and impact of study: B. Amyloliquefaciens (KJ461444) and P. pachastrellae (KM460937) inhibited the growth of P. capsici and F. solani thus reflecting the potential of the produced metabolites to be purified and used in combating plant pathogenic fungi.

Cloning, characterization and expression of a novel laccase gene Pclac2 from Phytophthora capsici

Laccases are blue copper oxidases (E.C. 1.10.3.2) that catalyze the one-electron oxidation of phenolics, aromatic amines, and other electron-rich substrates with the concomitant reduction of O2 to H2O. A novel laccase gene pclac2 and its corresponding full-length cDNA were cloned and characterized from Phytophthora capsici for the first time. The 1683 bp full-length cDNA of pclac2 encoded a mature laccase protein containing 560 amino acids preceded by a signal peptide of 23 amino acids. The deduced protein sequence of PCLAC2 showed high similarity with other known fungal laccases and contained four copper-binding conserved domains of typical laccase protein. In order to achieve a high level secretion and full activity expression of PCLAC2, expression vector pPIC9K with the Pichia pastoris expression system was used. The recombinant PCLAC2 protein was purified and showed on SDS-PAGE as a single band with an apparent molecular weight ca. 68 kDa. The high activity of purified PCLAC2, 84 U/mL, at the seventh day induced with methanol, was observed with 2,2'-azino-di-(3-ethylbenzothialozin-6-sulfonic acid) (ABTS) as substrate. The optimum pH and temperature for ABTS were 4.0 and 30 ºC, respectively . The reported data add a new piece to the knowledge about P. Capsici laccase multigene family and shed light on potential function about biotechnological and industrial applications of the individual laccase isoforms in oomycetes.

Estimation of Genetic Variation of Korean Isolates of Phytophthora capsici by Using Molecular Markers

Genetic diversity of 21 Korean Phytophthora capsici isolates was analyzed by using several molecular markers such as random amplified polymorphic DNA(RAPD), M-13, microsatellite and random amplified microsatellite sequences(RAMS). The overall average similarity coefficient among the isolates was 86% based on the combined data obtained by the molecular markers. No molecular markers were found to be associated with hosts or geographic regions. In addition to RAPD, analysis based on repeated sequences such as (GTG)5, M-13 and RAMS could be used to assess population structure of P. capsici.

A Rapid Radicle Assay for Prescreening Antagonistic Bacteria Against Phytophthora capsici on Pepper

A rapid radicle assay for prescreening antagonistic bacteria to Phytophthora capsici, causal agent of Phytophthora blight of pepper was developed. Sixty-four bacterial strains with in vitro antifungal activity selected out of 1,400 strains isolated from soils of Ansung, Chunan, Koyang, and Paju, Korea in 1998 were used for development of the bioassay. Uniformly germinated pepper seeds dipped in bacterial cells for 3 hours were placed near the edges of growing mycelia of P. capsici on water agar containing 0.02% glucose. Five-week-old pepper plants (cv. Nockwang) were inoculated to compare with results of the radicle assay developed in this study. For plant inoculation, pepper seeds were sown in potting mixtures incorporated with the bacterial strains, then transplanted into steam-sterilized soils 3 weeks later. Plants were hole-inoculated with zoospores of P. capsici 2 weeks after transplanting. Disease incidence and severity were determined in radicle and plant assessments, respectively. In radicle assay, six strains, GK-B15, GK-B25, OA-B26, OA-B36, PK-B09, and VK-B14 consistently showed the significant (P=0.05) disease reduction against radicle infection by the fungus, four of which also did in plant assessments. Strains OA-B36 and GK-B15 consistently reduced the fungal infection in both the radicle assay and the plant assessment. Therefore, prescreening strains using the radicle assay developed in this study followed by plant assay could reduce time and labor, and improved the possibility of selecting antagonistic bacteria for control of Phytophthora blight of peppers.

Electrophoretic protein patterns of three species of Phytophthora associated with black pod disease of cocoa (Theobroma cacao L.).

When electrophoretic profiles of native proteins from vegetative mycelia of Phytophthora palmivora, Phytophthora capsici and Phytophthora citrophthora causing black pod disease of cocoa in India were compared on a single Polyacrylamide gel, the isolates of same species were readily distinguished both qualitatively by visual similarity in banding patterns and quantitatively by calculating similarity coefficients. Similarity coefficients were generally much higher between isolates within a species than between isolates of different species. The dendrograms obtained after unweighted pair grouping with arithmetic averaging cluster analysis, revealed that all the isolates of Phytophthora capsici were highly homogenous and formed a single cluster. The isolates of Phytophthora citrophthora were resolved into two electrophoretic types which were clustered into two distinct sub groups. Phytophthora palmivora formed a separate group. Thus, the results reveal that polyacrylamide gel electrophoresis can be used successfully in distinguishing species and sub groups within a species of Phytophthora encountered on cocoa.