Paenibacillus Polymyxa E681 Research Articles

Influence of phenotypic variation of Paenibacillus polymyxa E681 on growth promotion in cucumbers.

The goal of the current study is to better understand how bacteria may adapt to survive under adverse environmental conditions by altering and improving their phenotypes. In this study, we report the consequences of phenotypic variation in Paenibacillus polymyxa E681 (E681), a plant growth-promoting rhizobacterium (PGPR), isolated from winter barley root that has a variety of advantageous effects on crop plants. In our previous study, two different types of bacterial cells in E681 were distinguished. We used the term F-type for the variant that doesn't produce endospores and B-type for the endospore-producing wild type. Under the circumstances of our experiment, the cucumber rhizosphere soil and the surface of the seeds produced phenotypic variance. On tryptic soy agar (TSA) plates, the B-type spontaneously converted into the F-type, but the reverse was not reversible. Intriguingly, the plant growth promotion test displayed that cucumber seedlings treated with F-type cells had characteristics resembling those of the untreated control. Whereas, growth promotion of cucumber seedlings treated with B-type depends on temperature conditions. In particular, an increased growth promotion was observed at a low temperature of 20°C. The phenotypic change from B-type to F-type did not occur at 20°C for 6 days in the growth curve analysis of E681, but it did occur on the fourth and second days at 30 and 37°C, respectively. Therefore, before using PGPR strains as a bacterial inoculant for sustainable agriculture, it is imperative to resolve phenotypic variance in these strains.

Occurrence of phenotypic variation in Paenibacillus polymyxa E681 associated with sporulation and carbohydrate metabolism

We report the phenotypic variation in Paenibacillus polymyxa E681 (E681), a plant growth-promoting rhizobacterium (PGPR) isolated from a winter barley root in Korea. Phenotypic variation (F-type) occurred when E681 (B-type) was grown in the media, and F-type was generated from B-type. B- and F-types were characterized by their morphological, Biolog, and GC-MIDI analyses. F-type cells altered the original biological capacity of B-type cells on endospore and flagella formation, changes in pH in culture, and carbon utilization. In growth curve analysis, B-type variants recovered bacterial growth as the variation occurred after the decline phase, but F-type variants did not. To determine this cause, we conducted comparative proteome analysis between B- and F-types using two-dimensional gel electrophoresis (2-DE). Of the identified proteins, 47% were involved in glycolysis and other metabolic pathways associated with carbohydrate metabolism. Therefore, our findings provide new knowledge on the mechanism of phenotypic variation and insights into agricultural biotechnology.

Cytosine Base Editor-Mediated Multiplex Genome Editing to Accelerate Discovery of Novel Antibiotics in Bacillus subtilis and Paenibacillus polymyxa.

Genome-based identification of new antibiotics is emerging as an alternative to traditional methods. However, uncovering hidden antibiotics under the background of known antibiotics remains a challenge. To over this problem using a quick and effective genetic approach, we developed a multiplex genome editing system using a cytosine base editor (CBE). The CBE system achieved simultaneous double, triple, quadruple, and quintuple gene editing with efficiencies of 100, 100, 83, and 75%, respectively, as well as the 100% editing efficiency of single targets in Bacillus subtilis. Whole-genome sequencing of the edited strains showed that they had an average of 8.5 off-target single-nucleotide variants at gRNA-independent positions. The CBE system was used to simultaneously knockout five known antibiotic biosynthetic gene clusters to leave only an uncharacterized polyketide biosynthetic gene cluster in Paenibacillus polymyxa E681. The polyketide showed antimicrobial activities against gram-positive bacteria, but not gram-negative bacteria and fungi. Therefore, our findings suggested that the CBE system might serve as a powerful tool for multiplex genome editing and greatly accelerating the unraveling of hidden antibiotics in Bacillus and Paenibacillus species.

Chronicle of a Soil Bacterium: Paenibacillus polymyxa E681 as a Tiny Guardian of Plant and Human Health.

The Gram-positive rhizosphere bacterium Paenibacillus polymyxa promotes plant growth and produces various antibiotics. Herein, we review research on this species over the past two and a half decades, and focus on the mechanisms of P. polymyxa strain E681, isolated from barley roots in the South Korea in 1995. Strain E681 has outstanding growth-promoting effects on barley, cucumber, pepper, sesame, and Arabidopsis thaliana and produces antimicrobial compounds that protect plants against pathogenic fungi, oomycetes, and bacteria. Induced systemic resistance elicited by treating seeds or roots with strain E681 is a possible mechanism for protecting systemic plant tissues from biotic and other environmental stresses. Genome sequencing has broadened our horizons for antibiotic development and other industrial applications beyond agricultural use. At least six gene clusters for the biosynthesis of antibiotics have been discovered, including polymyxin (pmx), which was recently re-instated as an antibiotic of last resort against Gram-negative drug-resistant bacteria. Three groups of antibiotic synthetases include the gene clusters that encode one for the non-ribosomal peptide polymyxin, fusaricidin, and tridecaptin, another for the lantibiotic paenilan, and the third for a polyketide. We successfully introduced the pmx gene cluster into the surrogate host Bacillus subtilis and created polymyxin derivatives by domain swapping. Furthermore, various E681 derivatives, including a high fusaricidin producer and strains lacking multi-antibiotics production, have been constructed by random mutagenesis and genome engineering. Thus, E681 is an important bacterium that contributes to both plant and human health.

Identification of the biosynthesis gene cluster for the novel lantibiotic paenilan from Paenibacillus polymyxa E681 and characterization of its product.

To analyse the function of a putative lantibiotic gene cluster of Paenibacillus polymyxa E681 and to characterize its product, paenilan. Comparative analysis of the lantibiotic gene cluster of E681 revealed that the cluster, consisting of 11 open reading frames, is involved in the biosynthesis of a class I lantibiotic. The pnlA gene encoding the prepeptide PnlA was identified and P. polymyxa strain EPT14 producing only paenilan was constructed by knockout of the other five antibiotic biosynthetic gene clusters of E681. Paenilan was purified from EPT14 culture by solvent partitioning, ODS silica gel chromatography and reversed-phase preparative HPLC. The molecular mass (2510·10Da) and structure of paenilan analysed by Nanoelectrospray ionization mass spectrometry (MS) and MS/MS showed that paenilan is a novel class I lantibiotic. Paenilan exhibited antimicrobial activity against Gram-positive bacteria such as Bacillus cereus, Micrococcus luteus and Paenibacillus durus. The paenilan gene is well-conserved in different Paenibacillus sp. isolated from globally distant places. The lantibiotic gene cluster of P. polymyxa E681 was analysed and its product, a novel and useful lantibiotic named paenilan that inhibits the growth of some Gram-positive bacteria, was characterized. Paenibacillus species are a good source of new lantibiotics, and the conservation of the paenilan gene among Paenibacillus sp. implies paenilan has an important function(s) for their survival.

Proteomic analyses of the interaction between the plant-growth promoting rhizobacterium Paenibacillus polymyxa E681 and Arabidopsis thaliana.

Plant growth-promoting rhizobacteria (PGPR) facilitate the plant growth and enhance their induced systemic resistance (ISR) against a variety of environmental stresses. In this study, we carried out integrative analyses on the proteome, transcriptome, and metabolome to investigate Arabidopsis root and shoot responses to the well-known PGPR strain Paenibacillus polymyxa (P. polymyxa) E681. Shoot fresh and root dry weights were increased, whereas root length was decreased by treatment with P. polymyxa E681. 2DE approach in conjunction with MALDI-TOF/TOF analysis revealed a total of 41 (17 spots in root, 24 spots in shoot) that were differentially expressed in response to P. polymyxa E681. Biological process- and molecular function-based bioinformatics analysis resulted in their classification into seven different protein groups. Of these, 36 proteins including amino acid metabolism, antioxidant, defense and stress response, photosynthesis, and plant hormone-related proteins were up-regulated, whereas five proteins including three carbohydrate metabolism- and one amino acid metabolism-related, and one unknown protein were down-regulated, respectively. A good correlation was observed between protein and transcript abundances for the 12 differentially expressed proteins during interactions as determined by qPCR analysis. Metabolite analysis using LC-MS/MS revealed highly increased levels of tryptophan, indole-3-acetonitrile (IAN), indole-3-acetic acid (IAA), and camalexin in the treated plants. Arabidopsis plant inoculated P. polymyxa E681 also showed resistance to Botrytis cinerea infection. Taken together these results suggest that P. polymyxa E681 may promote plant growth by induced metabolism and activation of defense-related proteins against fungal pathogen.

Root Exudation by Aphid Leaf Infestation Recruits Root-Associated Paenibacillus spp. to Lead Plant Insect Susceptibility.

Aphids are a large group of hemipteran pests that affect the physiology, growth, and development of plants by using piercing mouthparts to consume fluids from the host. Based an recent data, aphids modulate the microbiomes of plants and thereby affect the overall outcome of the biological interaction. However, in a few reports, aboveground aphids manipulate the metabolism of the host and facilitate infestations by rhizosphere bacteria (rhizobacteria). In this study, we evaluated whether aphids alter the plant resistance that is mediated by the bacterial community of the root system. The rhizobacteria were affected by aphid infestation of pepper, and a large population of gram-positive bacteria was detected. Notably, Paenibacillus spp. were the unique gram-positive bacteria to respond to changes induced by the aphids. Paenibacillus polymyxa E681 was used as a rhizobacterium model to assess the recruitment of bacteria to the rhizosphere by the phloem-sucking of aphids and to test the effect of P. polymyxa on the susceptibility of plants to aphids. The root exudates secreted from peppers infested with aphids increased the growth rate of P. polymyxa E681. The application of P. polymyxa E681 to pepper roots promoted the colonization of aphids within 2 days of inoculation. Collectively, our results suggest that aphid infestation modulated the root exudation, which led to the recruitment of rhizobacteria that manipulated the resistance of peppers to aphids. In this study, new information is provided on how the infestation of insects is facilitated through insect-derived modulation of plant resistance with the attraction of gram-positive rhizobacteria.

Biosynthesis of Polymyxins B, E, and P Using Genetically Engineered Polymyxin Synthetases in the Surrogate Host Bacillus subtilis.

The development of diverse polymyxin derivatives is needed to solve the toxicity and resistance problems of polymyxins. However, no platform has generated polymyxin derivatives by genetically engineering a polymyxin synthetase, which is a nonribosomal peptide synthetase. In this study, we present a two-step approach for the construction of engineered polymyxin synthetases by substituting the adenylation (A) domains of polymyxin A synthetase, which is encoded by the pmxABCDE gene cluster of Paenibacillus polymyxa E681. First, the seventh L-threonine-specific A-domain region in pmxA was substituted with the Lleucine- specific A-domain region obtained from P. polymyxa ATCC21830 to make polymyxin E synthetase, and then the sixth D-leucine-specific A-domain region (A6-D-Leu-domain) was substituted with the D-phenylalanine-specific A-domain region (A6-D-Phe-domain) obtained from P. polymyxa F4 to make polymyxin B synthetase. This step was performed in Escherichia coli on a pmxA-containing fosmid, using the lambda Red recombination system and the sacB gene as a counter-selectable marker. Next, the modified pmxA gene was fused to pmxBCDE on the chromosome of Bacillus subtilis BSK4dA, and the resulting recombinant strains BSK4-PB and BSK4-PE were confirmed to produce polymyxins B and E, respectively. We also succeeded in constructing the B. subtilis BSK4-PP strain, which produces polymyxin P, by singly substituting the A6-D-Leu-domain with the A6-D-Phe-domain. This is the first report in which polymyxin derivatives were generated by genetically engineering polymyxin synthetases. The two recombinant B. subtilis strains will be useful for improving the commercial production of polymyxins B and E, and they will facilitate the generation of novel polymyxin derivatives.

The effects of Paenibacillus polymyxa E681 on antifungal and crack remediation of cement paste.

This study investigated the antifungal effects of cement paste containing Paenibacillus polymyxa E681 against Aspergillus niger, a deleterious fungus commonly found in cement buildings and structures. To test the antifungal effects, cement paste containing P. polymyxa E681 was neutralized by CO2 gas, and the fungal growth inhibition was examined according to the clear zone around the cement specimen. In addition to the antifungal effects of the cement paste added with bacteria, calcium crystal precipitation of P. polymyxa E681 was examined by qualitative and quantitative analyses. The cement paste containing P. polymyxa E681 showed strong antifungal effects but fusA mutant (deficient in fusaricidin synthesis) showed no antifungal activity. Crack sealing of the cement paste treated with P. polymyxa E681 was captured by light microscope showed fungal growth inhibition and crack repairing in cement paste.

An antibiotic fusaricidin: a cyclic depsipeptide from Paenibacillus polymyxa E681 induces systemic resistance against Phytophthora blight of red-pepper

In this study fusaricidin, a cyclic depsipeptide isolated from Paenibacillus polymyxa E681 (E681), was demonstrated to control Phytophthora blight infection caused by Phytophthora capsici in red-pepper. The minimal inhibitory concentration (MIC) of fusaricidin was found to be 16 ppm against P. capsici. The disease severity of P. capsici was 40% at 0.1 ppm of fusaricidin when compared with water-treated control (81.7%) on post-treatment, whereas the disease severities on pre-treatment were 45% and 83.3% in fusaricidin (0.1 ppm) and water-treated control, respectively, in red-pepper plants. Significant (P < 0.05) disease suppression was observed on treatment with fusaricidin (0.1 ppm) by foliar spray and soil drench. The disease severity was drastically reduced to 3.3% by soil drench of fusaricidin (1.0 ppm), whereas in water-treated control, the disease severity was 83.3% in the first experiment. Fusaricidin at 0.1 ppm reduced disease severity of P. capsici to 27.5% when compared with positive control (43.1%) and water-treated control (66.2%) in the second experiment. Soft rot disease in tobacco was suppressed upon treatment with fusaricidin at 1.0 ppm by leaf infiltration. RT-PCR analyses of Arabidopsis thaliana revealed that there was an up-regulation of pathogenesis-related (PR) gene expression in wild type A. thaliana (Col-0), while there was no accumulation of PR genes, which implies that the mechanism of protection might be based on a salicylic acid-dependent pathway. This is the first report that fusaricidin exhibits protection against plant pathogens in addition to activity as an antibiotic agent. Hence, E681 can play a role in plant protection by secretion of ISR elicitors including fusaricidin.

Induced Resistance by a Long-Chain Bacterial Volatile: Elicitation of Plant Systemic Defense by a C13 Volatile Produced by Paenibacillus polymyxa

BackgroundSome strains of plant growth-promoting rhizobacteria (PGPR) elicit induced systemic resistance (ISR) by emission of volatile organic compounds (VOCs) including short chain alcohols, acetoin, and 2,3-butanediol. The objective of this study was to evaluate whether species-specific VOCs from PGPR strain Paenibacillus polymyxa E681 can promote growth and induce resistance in Arabidopsis.Methodology/Principal FindingsThe efficacy of induction was strain-specific, with stronger protection against Pseudomonas syringae pv. maculicola ES4326 in plants exposed to VOCs from P. polymyxa E681 versus Arabidopsis plants exposed to VOCs from a reference strain Bacillus subtilis GB03, which was previously shown to elicit ISR and plant growth promotion. VOC emissions released from E681 primed transcriptional expression of the salicylic acid, jasmonic acid, and ethylene signaling marker genes PR1, ChiB, and VSP2, respectively. In addition, strain E681 produced more than thirty low molecular-weight VOCs, of which tridecane was only produced by E681 and not found in GB03 or IN937a volatile blends. These strain-specific VOCs induced PR1 and VSP2 genes.Conclusions/SignificanceThese results provide new insight into the existence of a long chain VOC signaling molecule produced by P. polymyxa that can serve as a bacterial trigger of induced systemic resistance in planta.

Biological control and plant growth promoting capacity of rhizobacteria on pepper under greenhouse and field conditions

Plant growth promoting rhizobacteria Ochrobactrum lupini KUDC1013 and Novosphingobium pentaromativorans KUDC1065 isolated from Dokdo Island, S. Korea are capable of eliciting induced systemic resistance (ISR) in pepper against bacterial spot disease. The present study aimed to determine whether plant growth-promoting rhizobacteria (PGPR) strains including strain KUDC1013, strain KUDC1065, and Paenibacillus polymyxa E681 either singly or in combinations were evaluated to have the capacity for potential biological control and plant growth promotion effect in the field trials. Under greenhouse conditions, the induced systemic resistance (ISR) effect of treatment with strains KUDC1013 and KUDC1065 differed according to pepper growth stages. Drenching of 3-week-old pepper seedlings with the KUDC-1013 strain significantly reduced the disease symptoms. In contrast, treatment with the KUDC1065 strain significantly protected 5-week-old pepper seedlings. Under field conditions, peppers treated with PGPR mixtures containing E681 and KUDC1013, either in a two-way combination, were showed greater effect on plant growth than those treated with an individual treatment. Collectively, the application of mixtures of PGPR strains on pepper might be considered as a potential biological control under greenhouse and field conditions.

Characterization of sporulation histidine kinases of Paenibacillus polymyxa

Sporulation histidine kinases, which sense sporulation-specific signals and initiate phosphorelay reactions, are poorly conserved among Bacillus species. We found several putative genes for sporulation histidine kinases in the genome sequence of Paenibacillus polymyxa E681 and assayed the genes for complementation of sporulation mutants of Bacillus subtilis. One of these genes, Kin1377, significantly restored the sporulation deficiency of kinA kinB double mutant of B. subtilis, but not of B. subtilis spo0B mutant. These results indicated that Kin1377 requires B. subtilis Spo0B and possibly Spo0F to transfer phosphate to B. subtilis Spo0A. Another putative kinase, Kin1038, slightly restored the sporulation deficiencies of both kinA kinB double mutant and spo0B mutant of B. subtilis. However the sporulation deficiency of the B. subtilis spo0B mutant was significantly restored in the presence of both Kin1038 and P. polymyxa Spo0A. These results indicate that the overexpressed Kin1038 is able to interact directly with and activate P. polymyxa Spo0A, and that Spo0A can support spore formation in B. subtilis.

Isolation and Characterization of Transposon-Insertional Mutants from Paenibacillus polymyxa E681 Altering the Biosynthesis of Indole-3-Acetic Acid

We screened a mini-Tn10 insertional mutant library of the spore-forming bacterium Paenibacillus polymyxa E681 with variable indole-3-acetic acid (IAA) productivity. Four mutants, of which two showed a decrease in IAA production and the other two showed an increase in IAA production, were finally selected. Further analyses demonstrated different levels of IAA intermediates from culture supernatant of wild-type strain and mutants. In addition, mutants showed different promotions on the early growth of 10-day-old maize in terms of the increase in shoot and root weights. DNA fragments flanking the transposon insertion in four mutants were cloned and sequenced. The target sites of insertion were gene gpr1, disrupted at two sites, 49 bp downstream of the spo0F gene, and relA/spoT homologue, which codes for GPR1/FUN34/YaaH family protein, stage 0 sporulation protein F, and RelA/SpoT domain protein, respectively. This evidence suggests that there may be a number of genes involved in the regulation of IAA biosynthesis of P. polymyxa.

Improvement of biological control capacity of Paenibacillus polymyxa E681 by seed pelleting on sesame

Sesame is an important vegetable crop for the production of oil in Korea. The main obstacle of sesame cultivation is the occurrence of damping-off diseases and wilt caused by a complex of soil-borne pathogens in fields cultivated for two or more successive years. To protect sesame seedlings against these diseases, Paenibacillus polymyxa E681, a plant growth-promoting rhizobacterium (PGPR) previously shown to suppress disease incidence and promote growth on cucumber and pepper in the greenhouse and field experiments, was evaluated for its capacity for biological control and growth promotion in vitro and in situ. Seed treatment with strain E681 alone did not show consistent protection. Therefore, seed pelleting with strain E681 was attempted to increase the seed size and improve the stability and effectiveness of biocontrol capacity by strain E681. Through screening of pelleting materials, a combination of clay and vermiculite was selected for further experiments to enhance seed germination and root colonization of strain E681 on sesame. In greenhouse trials, formulations of strain E681 reduced disease incidence in disease-conducive soil. In the field, pelleting of sesame seeds with strain E681 significantly reduced pre- and post-emergence damping-off compared to the non-treated or pelleting alone controls; pelleting also promoted the plant growth and the grain yield. Furthermore, the efficacy of strain E681 for biological control and plant growth promotion was improved by sesame seed pelleting compared to the treatment with strain E681 alone. Hence, the application of strain E681 via seed pelleting offers potential to overcome some of the problems associated with successive years of sesame cultivation.

Study of mechanisms for plant growth promotion elicited by rhizobacteria in Arabidopsis thaliana

Plant growth-promoting rhizobacteria (PGPR) colonize plant roots and exert beneficial effects on plant health and development. We are investigating the mechanisms by which PGPR elicit plant growth promotion from the viewpoint of signal transduction pathways within plants. We report here our first study to determine if well-characterized PGPR strains, which previously demonstrated growth promotion of various other plants, also enhance plant growth in Arabidopsis thaliana. Eight different PGPR strains, including Bacillus subtilis GB03, B. amyloliquefaciens IN937a, B. pumilus SE-34, B. pumilus T4, B. pasteurii C9, Paenibacillus polymyxa E681, Pseudomonas fluorescens 89B-61, and Serratia marcescens 90-166, were evaluated for elicitation of growth promotion of wild-type and mutant Arabidopsis in vitro and in vivo. In vitro testing on MS medium indicated that all eight PGPR strains increased foliar fresh weight of Arabidopsis at distances of 2, 4, and 6 cm from the site of bacterial inoculation. Among the eight strains, IN937a and GB03 inhibited growth of Arabidopsis plants when the bacteria were inoculated 2 cm from the plants, while they significantly increased plant growth when inoculated 6 cm from the plants, suggesting that a bacterial metabolite that diffused into the agar accounted for growth promotion with this strain. In vivo, eight PGPR strains promoted foliar fresh weight under greenhouse conditions 4 weeks after sowing. To define signal transduction pathways associated with growth promotion elicited by PGPR, various plant-hormone mutants of Arabidopsis were evaluated in vitro and in vivo. Elicitation of growth promotion by PGPR strains in vitro involved signaling of brassinosteroid, IAA, salicylic acid, and gibberellins. In vivo testing indicated that ethylene signaling was involved in growth promotion. Results suggest that elicitation of growth promotion by PGPR in Arabidopsis is associated with several different signal transduction pathways and that such signaling may be different for plants grown in vitro vs. in vivo.

Colonization and Population Changes of a Biocontrol Agent, Paenibacillus polymyxa E681, in Seeds and Roots

Paenibacillus polymyxa E681, with its plant growth promotion and root colonization ability, has been proven to be a promising biocontrol agent of cucumber and barley. This study investigated the attributes related to the movement of bacteria from the seed to the radicle and to the whole root system. It also illustrated the existing form and population changes of the bacteria on seed and root using the scanning electron microscope and confocal laser scanning microscopy. The bacteria invaded and colonized the inside of the seed coat while the seeds were soaked in bacterial suspension. Almost the same number of bacteria on seed surface invaded the inside of the seed coat right after seed soaking. The population densities of E681 increased greatly inside as well as on the surface of the seed before the radicle emerged. The bacteria attached on the emerging radicle directly affected the initial population of newly emerg-ing root. The colonized cells on the root were arranged linearly toward the elongation of the root axis. In addition to colonizing the root surface, strain E681 was found inside the roots, where cells colonized the inter-cellular space between certain epidermal and cortical cells. When the cucumber seeds were soaked in bacterial suspension and sown in pot, the bacterial populations attached on both the surface and inside of the root were sustained up to harvesting time. This means that E681 successfully colonized the root of cucumber and sustained its population density up to harvesting time through seed treatment.

Ecologically Based Approaches to Management of Phytophthora Blight on Bell Pepper.

Phytophthora blight, caused by the oomycete pathogen, Phytophthora capsici, is a devastating disease on bell pepper and cucurbit crops in the United States and worldwide (29,40). P. capsici causes a root and crown rot, as well as an aerial blight of leaves, fruit, and stems, on bell pepper (Capsicum annuum), tomatoes, cucumber, watermelon, squash, and pumpkin (29,35, 40,57,73). The disease was first described on bell pepper in New Mexico in 1922 (40). In recent years, epidemics have been severe in areas of North Carolina, Florida, Georgia, Michigan, and New Jersey. Oospores are believed to provide the initial source of inoculum in the field, and the disease is polycyclic within seasons (1,7,59,60,67). In this article, we discuss the biology and epidemiology of Phytophthora blight on bell pepper and also describe management strategies that can be implemented based on existing knowledge of the ecology of this devastating pathogen. The objectives of ecologically based pest management (EBPM) are the safe, profitable, and durable management of pests that includes a total systems approach (25). EBPM relies primarily on biological input of knowledge concerning a pathogen life cycle, and secondarily, when necessary, on physical, chemical, and biological supplements for disease management. An understanding of the ecological processes that are suppressive to plant diseases is emphasized rather than secondary inputs (25). Fortunately, we have a considerable amount of information available on the biology and ecology of P. capsici, which can now be integrated to improve our ability to manage the disease using ecologically based approaches. Strategies recommended for management of Phytophthora blight involve integrated approaches that focus first on cultural practices that reduce high soil moisture conditions, but also include monitoring and reduction of propagules of P. capsici that persist in the soil, utilization of cultivars with resistance to the disease, and when necessary, judicious fungicide applications. Symptoms and Life Cycle P. capsici can infect virtually every part of the pepper plant. The pathogen causes a root and crown rot on pepper (Fig. 1) and also forms distinctive black lesions on the stem (Fig. 2). P. capsici can also infect the leaves and causes lesions that are circular, grayish brown, and water-soaked (Fig. 3). Leaf lesions and stem lesions are common when inoculum is splash dispersed from the soil to lower portions of the plant. The pathogen can also infect fruit and causes lesions that are typically covered with white sporangia, a sign of the pathogen (Fig. 4). P. capsici typically causes a fruit rot or stem rot on cucumbers and squash (Fig. 5). P. capsici reproduces by both sexual and asexual means (Fig. 6). The pathogen produces two mating types, known as the A1 and A2. These are actually compatibility types and do not correspond to dimorphic forms. Each mating type produces hormones that are responsible for gametangia differentiation in the opposite mating type. Both A1 and A2 mating types are common in fields in North Carolina and have also been identified within the same plant (59). P. capsici produces a male gametangium, called the antheridium, and a female gametangium, called the oogonium. The antheridium is amphigynous in this species. Meiosis occurs within the gametangia, and plasmogamy and karyogamy result