Segregation following interspecific transfer of isolated nuclei between Phytophthora parasitica and P. capsici

Nuclei isolated from metalaxyl-resistant (MR) protoplasts of Phytophthora parasitica were transferred into chloroneb-resistant (CnR) protoplasts of Phytophthora capsici and vice versa, with an average success rate of 2.6 x 10(-4) (protoplasts with donor nuclei/regenerated protoplasts), using a selective medium containing only the fungicide tolerated by the nuclear donor. No colonies appeared when self-fusion products of donor nuclei or recipient protoplasts were exposed to the selective medium. Colonies produced by the nuclear transfer formed sectors commonly, and differed from the parental types in appearance. All the zoospores produced by the nuclear hybrids were of normal size, and one-fifth of them contained both MR and CnR genes. Since zoospores are mostly uninucleate, these results indicated the occurrence of chromosome re-assortment or mitotic crossing-over following the production of transitory tetraploids, followed by diploidization during zoosporogenesis, thus suggesting the completion of events leading to a parasexual cycle. Hyphal fragment cultures from a nuclear hybrid tested showed considerable variation in growth rate, mycelial morphology, and level of resistance to metalaxyl, indicating uneven distribution and continuous segregation of different types of nuclei in mycelia during vegetative growth.     

Genetic diversity of Phytophthora capsici isolates from pepper and pumpkin in Argentina

Phytophthora capsici is a soilborne oomycete plant pathogen that limits pepper production worldwide. The population structure varies significantly depending on the location (e.g. Peru vs. USA) and little is known about the diversity of P. capsici in Argentina. Our objective was to assess the diversity of P. capsici in Argentina at key pepper production areas. Forty isolates were recovered 2006-2009 from pepper and one isolate from pumpkin at 11 locations. Isolates were assessed for mating type, mefenoxam sensitivity and multilocus single nucleotide polymorphism (SNP) genotype profiles. Ten isolates with identical SNP profiles also were genotyped with amplified fragment length polymorphism (AFLP) markers. All 41 isolates had the A1 mating type and were sensitive to mefenoxam. Genotypic analysis using eight polymorphic SNP markers indicated 87% of the isolates had the same multilocus genotype, which is fixed for heterozygosity at seven of the eight SNP sites. AFLP analyses confirmed these findings, and overall it appears that clonal reproduction drives the population structure of P. capsici in Argentina. The implications for breeding resistant peppers and overall disease management are discussed.     

Paenibacillus polymyxa antagonizes oomycete plant pathogens Phytophthora palmivora and Pythium aphanidermatum

Aim:  To find sustainable alternatives to the application of synthetic chemicals for oomycete pathogen suppression.
Methods and Results:  Here, we present experiments on an Arabidopsis thaliana model system in which we studied the antagonistic properties of rhizobacterium Paenibacillus polymyxa strains towards the oomycete plant pathogens Phytophthora palmivora and Pythium aphanidermatum . We carried out studies on agar plates, in liquid media and in soil. Our results indicate that P. polymyxa strains significantly reduced P. aphanidermatum and P. palmivora colonization in liquid assays. Most plants that had been treated with P. polymyxa survived the P. aphanidermatum inoculations in soil assays.
Conclusions:  The antagonistic abilities of both systems correlated well with mycoidal substance production and not with the production of antagonistic substances from the biocontrol bacteria.
Significance and Impact of the Study:  Our experiments highlight the need to take biofilm formation and niche exclusion mechanisms into consideration for biocontrol assays performed under natural conditions.     

Phytophthora parasitica biofilm formation: installation and organization of microcolonies on the surface of a host plant

Zoospores of the oomycete Phytophthora parasitica establish microbial spheroid microcolonies and biofilms on the surface of wounded leaves of their host, Nicotiana tabacum . The formation of microcolonies involves the movement of some zoospores towards attractants from wound sites, followed by their irreversible adsorption and the formation of a cluster of cells. These cells drive the migration of a second wave of zoospores (several hundreds cells) by setting up an external chemotactic gradient leading to massive zoospore encystment and cyst-orientated germination. Zoospores that are still swimming at this stage circulate within the nascent biofilm by opening channels. Concomitantly, the cell population secretes various substances to elaborate an extracellular mucilage. Embedded within the extracellular matrix, biofilm cells are organized into a structured community as coacervates. The granular surface is composed of individual cysts, located on the outside of the microcolony. Hyphae from these cysts plunge downwards towards the dense core formed by the founder cells. This report is the first to show the installation and organization of a biofilm formed by eukaryotic cells on plant surfaces. The P. parasitica microcolonies constitute heterogeneous microenvironments for the embedded and circulating cells. They may affect plant–pathogen interactions by serving as reservoirs for pathogenic microorganisms, as protecting niche against host defences or as structures for infecting populations.     

Retraction of flagella by Phytophthora parasitica var. nicotiana zoospores

Summary Light and electron microscope evidence is presented to show that retraction of flagella may occur in Phytophthora parasitica zoospores during encystment.     

Synthesis of beta-D-glucose oligosaccharides from Phytophthora parasitica

The glucohexaose, beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->6)]-beta-D-Glcp-(1-->3)-D-Glcp, was synthesized as its allyl glycoside via 3+3 strategy. The trisaccharide donor, 2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl-(1-->3)-2,4,6-tri-O-acetyl-beta-D-glucopyranosyl-(1-->3)-2,4,6-tri-O-acetyl-alpha-D-glucopyranosyl trichloroacetimidate (11), was obtained by 3-selective coupling of isopropyl 4,6-O-benzylidene-1-thio-beta-D-glucopyranoside (2) with 2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl-(1-->3)-2-O-acetyl-4,6-O-benzylidene-alpha-D-glucopyranosyl trichloroacetimidate (6), followed by hydrolysis, acetylation, dethiolation, and trichloroacetimidation. Meanwhile, the trisaccharide acceptor, allyl 2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl-(1-->3)-2-O-acetyl-beta-D-glucopyranosyl-(1-->3)-4,6-di-O-acetyl-2-O-benzoyl-alpha-D-glucopyranoside (14), was prepared by coupling of allyl 4,6-di-O-acetyl-2-O-benzoyl-alpha-D-glucopyranoside (12) with 6, followed by debenzylidenation. Condensation of 14 with 11, followed by deacylation, gave the target hexaoside. A beta-(1-->3)-linked tetrasaccharide 29 was also synthesized with methyl 2-O-benzoyl-4,6-O-benzylidene-beta-D-glucopyranosyl-(1-->3)-2,4,6-tri-O-acetyl-beta-D-glucopyranoside (25) as the acceptor and acylated beta-(1-->3)-linked disaccharide 21 as the donor.     

Characterization of elicitin-like phospholipases isolated from Phytophthora capsici culture filtrate.

The phytopathogenic oomycete Phytophthora capsici secretes in culture a phospholipase activity. Two enzyme isoforms exhibiting a high phospholipase B activity were isolated by chromatography and electrophoresis. They differ in their apparent molar masses (22 and 32 kDa). Both proteins are glycosylated and share the same N-terminal amino acid sequence up to the 39th residue with a high homology with capsicein, the P. capsici elicitin. Although devoid of phospholipase activity, capsicein was shown by circular dichroism to specifically interact with negatively charged phospholipids, suggesting that the membrane lipids could be a potential target for elicitins.     

Structural studies of the extracellular beta-D-glucans from Phytophthora parasitica Dastur

Several extracellular glucans have been isolated from Phytophthora parasitica Dastur, a phytopathogenic fungus of the carnation. These polysaccharides consist of a mixture of (1-->3)(1-->6)-beta-D-glucans whose molecular masses varied from 1 x 10(4) to 5 x 10(6) Da. All of these polysaccharides have a main chain of beta-(1-->3)-linked D-glucose residues substituted with mono-, di- and oligo-saccharidic chains attached through (1-->6) linkages.     

Isolation and activity of Xenorhabdus antimicrobial compounds against the plant pathogens Erwinia amylovora and Phytophthora nicotianae

Aims:  Broad-spectrum antibiotics produced by symbiotic bacteria [entomopathogenic bacterium (EPB)] of entomopathogenic nematodes keep monoxenic conditions in insect cadavers in soil. This study evaluated antibiotics produced by EPB for their potential to control plant pathogenic bacteria and oomycetes.
Methods and Results:  Entomopathogenic bacterium produce antibiotics effective against the fire blight bacterium Erwinia amylovora, including streptomycin resistant strains, and were as effective in phytotron experiments as kasugamycin or streptomycin. Xenorhabdus budapestensis and X . szentirmaii antibiotics inhibited colony formation and mycelial growth of Phytophthora nicotianae. From X . budapestensis, an arginine-rich fraction (bicornutin) was adsorbed by Amberlite^® XAD 1180, and eluted with methanol : 1  n HCI (99 : 1). Bicornutin inactivated zoospores, and inhibited germination and colony formation of cystospores at <<25 ppm. An UV-active molecule (bicornutin-A, MW = 826), separated by HPLC and thin-layer chromatography, was identified as a novel hexa-peptide : RLRRRX.
Conclusions:  Xenorhabdus budapestensis produces metabolites with strong antibacterial and cytotoxic activity. Individual compounds can be isolated, identified and patented, but their full antimicrobial potential may be multiplied by synergic interactions.
Significance and Impact of the Study:  Active compounds of two new Xenorhabdus species might control plant diseases caused by pathogens of great importance to agriculture such as Erw. amylovora and P . nicotianae .     

The effect of size and acetylation degree of chitosan derivatives on tobacco plant protection against Phytophthora parasitica nicotianae

Enzymatic degradation of chitosan polymer with Pectinex Ultra SPL was used to obtain derivatives with biological potential as protective agents against Phytophthora parasitica nicotianae (Ppn) in tobacco plants. The 24 h hydrolysate showed the highest Ppn antipathogenic activity and the chitosan native polymer the lowest. The in vitro growth inhibition of several Phytophthora parasitica strains by two chitosans of different DA was compared. While less acetylated chitosan (DA 1%) fully inhibited three P. parasitica strains at the doses 500 and 1000 mg/l the second polymer (DA 36.5%) never completely inhibited such strains. When comparing two polymers of similar molecular weight and different DA, again the highest antipathogenic activity was for the less acetylated polymer. However, degraded chitosan always showed the highest pathogen growth inhibition. Additionally, a bioassay in tobacco seedlings to test plant protection against Ppn by foliar application demonstrated that partially acetylated chitosan and its hydrolysate induced systemic resistance and higher levels of glucanase activity than less acetylated chitosan. Similarly, when treatments were applied as seeds coating before planting, about 46% of plant protection was obtained using chitosan hydrolysate. It was concluded that, while less acetylated and degraded chitosan are better for direct inhibition of pathogen growth, partially acetylated and degraded chitosan are suitable to protect tobacco against P. parasitica by systemic induction of plant resistance.     

辣椒疫霉PcCRN20-C蛋白的表达纯化及结晶

CRN(crinkling and necrosis-inducing protein)为疫霉菌在与寄主互作过程中分泌的一类特有胞质效应因子,干扰寄主细胞正常的生理代谢和功能。采用PCR法从辣椒疫霉LT1534菌株c DNA中克隆PcCRN20-C基因。该基因序列长783bp,编码261个氨基酸。构建重组表达载体,并转化大肠杆菌BL21(DE3)。在优化条件下诱导表达重组蛋白,利用Ni-NTA金属螯合层析、离子交换层析、分子筛层析和胰蛋白酶酶解技术获得高纯目的蛋白,SDS-PAGE分析表明,蛋白质分子量约为25kDa。采用座滴气相扩散法进行晶体制备和筛选,成功获得了蛋白质晶体,并通过X-射线衍射仪收集了晶体衍射花样。结合蛋白质晶体学方法,获得了有衍射的辣椒疫霉PcCRN20-C蛋白晶体,为进一步研究CRN蛋白的结构与病原菌致病机制提供参考资料。     

Production of Kestoses (Fructosylsucroses) by Phytophthora parasitica var. nicotianae

The synthesis of kestoses (trisaccharides composed of two fructose units and one glucose unit) by races 0 and 1 of Phytophthora parasitica var. nicotianae is shown. The trisaccharide is found in culture filtrates of isolates grown in liquid media containing 3% sucrose. The utilization of sucrose and trisaccharide formation by the organisms over a 16-day period is described. The kestoses were identified by chemical and enzymatic analysis, and two of three possible isomers were found.     

Genetic Diversity,Population Structure,and Resistance to Phytophthora capsici of a Worldwide Collection of Eggplant Germplasm

Eggplant (Solanum melongena L.) is an important solanaceous crop with high phenotypic diversity and moderate genotypic diversity. Ninety-nine genotypes of eggplant germplasm (species (S. melongena, S. incanum, S. linnaeanum and S. gilo), landraces and heirloom cultivars) from 32 countries and five continents were evaluated for genetic diversity, population structure, fruit shape, and disease resistance to Phytophthora fruit rot. Fruits from each line were measured for fruit shape and evaluated for resistance to two Phytophthora capsici isolates seven days post inoculation. Only one accession (PI 413784) was completely resistant to both isolates evaluated. Partial resistance to Phytophthora fruit rot was found in accessions from all four eggplant species evaluated in this study. Genetic diversity and population structure were assessed using 22 polymorphic simple sequence repeats (SSRs). The polymorphism information content (PIC) for the population was moderate (0.49) in the population. Genetic analyses using the program STRUCTURE indicated the existence of four genetic clusters within the eggplant collection. Population structure was detected when eggplant lines were grouped by species, continent of origin, country of origin, fruit shape and disease resistance.