Quantifying the relationship between disease severity and the amount of Aphanomyces euteiches detected in roots of alfalfa and pea with a real-time PCR assay

A real-time PCR assay was used to quantify the relationship in alfalfa and pea between disease severity and the amount of Aphanomyces euteiches detected in roots. The study included isolates of race 1 and race 2 of the alfalfa pathovar of A. euteiches and an isolate obtained from diseased pea. Spearman rank correlations between pathogen DNA content and disease severity index (DSI) ratings were positive ( ? 0.57) and significant (P  0.0007) for individual alfalfa plants, bulked alfalfa plant samples, and individual pea plants. In all experiments, significantly more pathogen was detected in susceptible populations than in resistant populations. The results clearly demonstrate that resistance to A. euteiches in both alfalfa and pea is characterized by a reduction in pathogen colonization relative to levels observed for susceptible reactions. The assay was very specific for A. euteiches, producing very linear assays with DNA extracted from pathogen isolates obtained from alfalfa, pea, and bean. Possible applications of the assay in conjunction with other real-time PCR assays specific to other legume pathogens are discussed in relation to simultaneous disease screening for multiple plant pathogens and the study of microbial population dynamics in mixed plant infections.     

Biological Control of Damping-Off of Alfalfa Seedlings with Bacillus cereus UW85

We explored the potential of biological control of alfalfa (Medicago sativa L.) seedling damping-off caused by Phytophthora megasperma f. sp. medicaginis by screening root-associated bacteria for disease suppression activity in a laboratory bioassay. A total of 700 bacterial strains were isolated from the roots of field-grown alfalfa plants by using Trypticase soy agar. A simple, rapid assay was developed to screen the bacteria for the ability to reduce the mortality of Iroquois alfalfa seedlings that were inoculated with P. megasperma f. sp. medicaginis zoospores. Two-day-old seedlings were planted in culture tubes containing moist vermiculite, and each tube was inoculated with a different bacterial culture. Sufficient P. megasperma f. sp. medicaginis zoospores were added to each tube to result in 100% mortality of control seedlings. Of the 700 bacterial isolates tested, only 1, which was identified as Bacillus cereus and designated UW85, reduced seedling mortality to 0% in the initial screen and in two secondary screens. Both fully sporulated cultures containing predominantly released spores and sterile filtrates of these cultures of UW85 were effective in protecting seedlings from damping-off; filtrates of cultures containing predominantly vegetative cells or endospores inside the parent cell had low biocontrol activity. Cultures grown in two semidefined media had significantly greater biocontrol activities than cultures grown in the complex tryptic soy medium. In a small-scale trial in a field infested with P. megasperma f. sp. medicaginis, coating seeds with UW85 significantly increased the emergence of alfalfa. The results suggest that UW85 may have potential as a biocontrol agent for alfalfa damping-off, thus providing an alternative to current disease control strategies.     

Biological Control of Phytophthora Root Rots on Alfalfa and Soybean with Streptomyces

A collection of 53 antibiotic-producing Streptomyces isolated from soils from Minnesota, Nebraska, and Washington were evaluated for their ability to inhibit plant pathogenic Phytophthora medicaginis and Phytophthora sojae in vitro. Eight isolates having the greatest pathogen-inhibitory capabilities were subsequently tested for their ability to control Phytophthora root rots on alfalfa and soybean in sterilized vermiculite and naturally infested field soil. The Streptomyces isolates tested significantly reduced root rot severity in alfalfa and soybean caused by P. medicaginis and P. sojae, respectively (P < 0.05). On alfalfa, isolates varied in their effect on plant disease severity, percentage dead plants, and plant biomass in the presence of the pathogen. The same eight isolates of Streptomyces were also tested for inhibitory activities against each other and against three strains of Bradyrhizobium japonicum and two strains of Sinorhizobium meliloti isolated from soybean and alfalfa, respectively. Streptomyces isolates clustered into two major compatibility groups: isolates within the same group were noninhibitory toward one another in vitro. The compatibility groups corresponded with groupings obtained based upon inhibition of B. japonicum and S. meliloti strains.     

Interactions between the vesicular-arbuscular mycorrhizal fungus Glomus fascicuhtum and Aphanomyces euteiches root rot of peas

Interactions between Glomus fasciculatum and Aphanomyces euteiches root rot of peas (Pisum sativum), were studied in pot experiments using irradiated soil. Infections with the pathogen were suppressed by VAM when plants were challenge inoculated after two weeks. No reduction of the pathogen was detected when the plants were inoculated with both fungi at the same time. The suppression of the pathogen, obtained by preinoculation with G. fasciculatum, was not reduced when the inoculum level of the pathogen was increased thirty times. The induced resistance to A. euteiches in VAM plants was partially a systemic effect. When root systems were split into two halves, one with mycorrhiza and one with A. euteiches, the oospore production was reduced in both root systems. The infection with the pathogen was only suppressed when both fungi were present in the same pot. The background for the induced resistance is discussed.     

Rapid detection of Phytophthora nicotianae by simple DNA extraction and real‐time loop‐mediated isothermal amplification assay

Phytophthora nicotianae is a phytopathogenic oomycete with a wide host range and worldwide distribution. Rapid detection and diagnosis at the early stages of disease development are important for the effective control of P. nicotianae. In this study, we designed a simple and rapid loop‐mediated isothermal amplification (LAMP)‐based detection method for P. nicotianae. We tested three DNA extraction methods and selected the Kaneka Easy DNA Extraction Kit version 2, which is rapid and robust for LAMP‐based detection. The designed primers were tested using mycelial DNA from 35 species (81 isolates) of Phytophthora, 12 species (12 isolates) of Pythium, one isolate of Phytopythium and one isolate each from seven other soil‐borne pathogens. All of the 42 P. nicotianae isolates were detected by these primers, and no other isolates gave positive results. Three isolates were tested for the sensitivity of the reaction, and the lowest amounts of template DNA that could be detected were 10 fg for two isolates and 1 fg for the third. The target was detected within 25 min in all tested samples, including DNA extracted from both inoculated and naturally infected plants. In contrast, PCR assays with P. nicotianae‐specific primers failed or showed weakened detection in several samples. Thus, we found that the rapid DNA extraction and LAMP assay methods developed in this study can be used to detect P. nicotianae with high sensitivity, specificity and stability.     

Proteomic Profiling Unravels Insights into the Molecular Background Underlying Increased <Emphasis Type="Italic">Aphanomyces euteiches</Emphasis>-Tolerance of <Emphasis Type="Italic">Medicago truncatula</Emphasis>

To investigate the molecular mechanisms underlying susceptibility of legumes to the root pathogen Aphanomyces euteiches (oomycota), comparative proteomic studies have been carried out. In a first approach, we have analysed two Medicago truncatula lines of the French CORE collection (F83.005-5 (R2002) and F83.005-9 (R2002)), which showed either increased or decreased susceptibility to A. euteiches as compared to the widely adopted line A17. Several proteins were identified to be differentially induced after pathogen challenge in the two M. truncatula accessions with altered disease susceptibility, whereof proteins with increased abundances in the more resistant line F83.005-9 could be involved in mechanisms that lead to an improved disease resistance. Among these proteins, we identified two proteasome alpha subunits, which might be involved in defense response. To broaden our studies on A. euteiches-tolerance of M. truncatula, we investigated two other phenomena that lead to an either increased A. euteiches-resistance or to an enhanced susceptibility. The topic of an enhanced plant resistance to A. euteiches was studied in plants showing a bioprotective effect of a pre-established arbuscular mycorrhiza (AM) symbiosis. Evaluation of root fresh weights and pathogen spreading in the root system clearly indicate that mycorrhizal plants show increased A. euteiches-resistance as compared to non-mycorrhizal plants. Proteome analyses revealed the induction of similar protein patterns as in the M. truncatula accessions with comparatively high resistance level to A. euteiches. In a third approach, increased A. euteiches susceptibility was effected by exogenous abscisic acid (ABA) application prior to root infection. Evaluation of the abundance levels of a group of pathogenesis related class 10 (PR10)-like proteins, which were previously identified to be regulated after A. euteiches infection, revealed a correlation between the abundance levels of these proteins and the A. euteiches infection level or severity. Requests concerning seeds from the Medicago truncatula lines F83.005-5 and F83.005-9 should be addressed to Jean-Marie Prospéri, INRA-SGAP Laboratory, Laboratoire de Ressources Génétiques et d’Amélioration des Luzernes méditerranéennes, Mauguio, France, jean-marie.prosperi@ensam.inra.fr.     

Enhanced resistance in Theobroma cacao against oomycete and fungal pathogens by secretion of phosphatidylinositol‐3‐phosphate‐binding proteins

The internalization of some oomycete and fungal pathogen effectors into host plant cells has been reported to be blocked by proteins that bind to the effectors' cell entry receptor, phosphatidylinositol‐3‐phosphate (PI3P). This finding suggested a novel strategy for disease control by engineering plants to secrete PI3P‐binding proteins. In this study, we tested this strategy using the chocolate tree Theobroma cacao. Transient expression and secretion of four different PI3P‐binding proteins in detached leaves of T. cacao greatly reduced infection by two oomycete pathogens, Phytophthora tropicalis and Phytophthora palmivora, which cause black pod disease. Lesion size and pathogen growth were reduced by up to 85%. Resistance was not conferred by proteins lacking a secretory leader, by proteins with mutations in their PI3P‐binding site, or by a secreted PI4P‐binding protein. Stably transformed, transgenic T. cacao plants expressing two different PI3P‐binding proteins showed substantially enhanced resistance to both P. tropicalis and P. palmivora, as well as to the fungal pathogen Colletotrichum theobromicola. These results demonstrate that secretion of PI3P‐binding proteins is an effective way to increase disease resistance in T. cacao, and potentially in other plants, against a broad spectrum of pathogens.     

The c‐di‐GMP phosphodiesterase BifA is involved in the virulence of bacteria from the Pseudomonas syringae complex

In a recent screen for novel virulence factors involved in the interaction between Pseudomonas savastanoi pv. savastanoi and the olive tree, a mutant was selected that contained a transposon insertion in a putative cyclic diguanylate (c‐di‐GMP) phosphodiesterase‐encoding gene. This gene displayed high similarity to bifA of Pseudomonas aeruginosa and Pseudomonas putida. Here, we examined the role of BifA in free‐living and virulence‐related phenotypes of two bacterial plant pathogens in the Pseudomonas syringae complex, the tumour‐inducing pathogen of woody hosts, P. savastanoi pv. savastanoi NCPPB 3335, and the pathogen of tomato and Arabidopsis, P. syringae pv. tomato DC3000. We showed that deletion of the bifA gene resulted in decreased swimming motility of both bacteria and inhibited swarming motility of DC3000. In contrast, overexpression of BifA in P. savastanoi pv. savastanoi had a positive impact on swimming motility and negatively affected biofilm formation. Deletion of bifA in NCPPB 3335 and DC3000 resulted in reduced fitness and virulence of the microbes in olive (NCPPB 3335) and tomato (DC3000) plants. In addition, real‐time monitoring of olive plants infected with green fluorescent protein (GFP)‐tagged P. savastanoi cells displayed an altered spatial distribution of mutant ΔbifA cells inside olive knots compared with the wild‐type strain. All free‐living phenotypes that were altered in both ΔbifA mutants, as well as the virulence of the NCPPB 3335 ΔbifA mutant in olive plants, were fully rescued by complementation with P. aeruginosa BifA, whose phosphodiesterase activity has been demonstrated. Thus, these results suggest that P. syringae and P. savastanoi BifA are also active phosphodiesterases. This first demonstration of the involvement of a putative phosphodiesterase in the virulence of the P. syringae complex provides confirmation of the role of c‐di‐GMP signalling in the virulence of this group of plant pathogens.     

MoNSTR‐seq,a restriction site‐associated DNA sequencing technique to characterize Agrobacterium‐mediated transfer‐DNA insertions in Phomopsis longicolla

Phomopsis longicolla (Hobbs) causes Phomopsis seed decay and stem lesions in soybean (Glycine max). In this study, a novel, high‐throughput adaptation of RAD‐seq termed MoNSTR‐seq (Mutation analysis via Next‐generation DNA Sequencing of T‐DNA Regions) was developed to determine the genomic location of T‐DNA insertions in P. longicolla mutants. Insertional mutants were created via Agrobacterium tumefaciens‐mediated transformation, and one mutant, strain PL343, was further investigated due to impaired stem lesion formation. Mutation analysis via Next‐generation DNA Sequencing of T‐DNA Regions, in which DNA libraries are created with two distinct restriction enzymes and customized adapters to simultaneously enrich both T‐DNA insertion borders, was developed to characterize the genomic lesion in strain PL343. MoNSTR‐seq successfully identified a T‐DNA insertion in the predicted promoter region of a gene encoding a cellobiose dehydrogenase (CDH1), and the position of the T‐DNA insertion in strain PL343 was confirmed by Sanger sequencing. Thus, MoNSTR‐seq represents an effective tool for molecular genetics in P. longicolla, and is readily adaptable for use in diverse fungal species.

Significance and Impact of the Study

This study describes MoNSTR‐seq (Mutation analysis via Next‐generation DNA Sequencing of T‐DNA Regions), an adaptation of restriction site‐associated DNA sequencing (RAD‐seq) to identify the position of transfer‐DNA (T‐DNA) insertions in the genome of Phomopsis longicolla, an important pathogen of soybean. The technique enables high‐throughput characterization of mutants generated via Agrobacterium tumefaciens‐mediated transformation (ATMT), thus accelerating gene discovery via forward genetics. This technique represents a significant advancement over existing approaches to characterize T‐DNA insertions in fungal genomes. With minor modifications, this technique could be easily adapted to taxonomically diverse fungal pathogens and additional mutagenesis cassettes.     

Species‐Specific PCR‐Based Assay for Identification and Detection of Phomopsis (Diaporthe) azadirachtae Causing Die‐Back Disease in Azadirachta indica

Die‐back disease caused by Phomopsis (Diaporthe) azadirachtae is the devastating disease of Azadirachta indica. Accurate identification of P. azadirachtae is always problematic due to morphological plasticity and delayed appearance of conidia. A species‐specific PCR‐based assay was developed for rapid and reliable identification of P. azadirachtae by designing a species‐specific primer‐targeting ITS region of P. azadirachtae isolates. The assay was validated with DNA isolated from different Phomopsis species and other fungal isolates. The PCR assay amplified 313‐bp product from all the isolates of P. azadirachtae and not from any other Phomopsis species or any genera indicating its specificity. The assay successfully detected the pathogen DNA in naturally and artificially infected neem seeds and twigs indicating its applicability in seed quarantine and seed health testing. The sensitivity of the assay was 100 fg when genomic DNA of all isolates was analysed. The PCR‐based assay was 92% effective in comparison with seed plating technique in detecting the pathogen. This is the first report on the development of species‐specific PCR assay for identification and detection of P. azadirachtae. Thus, PCR‐based assay developed is very specific, rapid, confirmatory and sensitive tool for detection of pathogen P. azadirachtae at early stages.     

Specific detection of Pectobacterium carotovorum by loop‐mediated isothermal amplification

Potatoes are an important agroeconomic crop worldwide and maceration diseases caused by pectolytic bacterial pathogens result in significant pre‐ and post‐harvest losses. Pectobacterium carotovorum shares a common host range with other Pectobacterium spp. and other members of the Enterobacteriaceae, such as Dickeya spp. As these pathogens cannot be clearly differentiated on the basis of the symptoms they cause, improved methods of identification are critical for the determination of sources of contamination. Current standardized methods for the differentiation of pectolytic species are time consuming and require trained personnel, as they rely on traditional bacteriological practices that do not always produce conclusive results. In this growing world market, there is a need for rapid diagnostic tests that can differentiate between pectolytic pathogens, as well as separate them from non‐pectolytic enteric bacteria associated with soft rots of potato. An assay has been designed previously to detect the temperate pathogen Pectobacterium atrosepticum, but there is currently no recognized rapid assay for the detection of the tropical/subtropical counterpart, Pectobacterium carotovorum. This report describes the development of a loop‐mediated isothermal amplification (LAMP) assay that detects P. carotovorum with high specificity. The assay was evaluated using all known species of Pectobacterium and only showed positive reactions for P. carotovorum. This assay was also tested against 15 non‐target genera of plant‐associated bacteria and did not produce any false positives. The LAMP assay described here can be used as a rapid test for the differentiation of P. carotovorum from other pectolytic pathogens, and its gene target can be the basis for the development of other molecular‐based detection assays.     

Vector within‐host feeding preference mediates transmission of a heterogeneously distributed pathogen

1. Ecological theory predicts that vector preference for certain host species or discrimination between infected versus uninfected hosts impacts disease incidence. However, little information exists on the extent to which vector within‐host feeding preference mediates transmission. This may be particularly important for plant pathogens, such as sharpshooter transmission of the bacterium Xylella fastidiosa, which are distributed irregularly throughout hosts. 2. We documented the within‐host distribution of two vector species that differ in transmission efficiency, the leafhoppers Draeculacephala minerva and Graphocephala atropunctata, and which are free to move throughout entirely caged alfalfa plants. The more efficient vector D. minerva fed preferentially at the base of the plant near the soil surface, whereas the less efficient G. atropunctata preferred overwhelming the top of the plant. 3. Next we documented X. fastidiosa heterogeneity in mechanically inoculated plants. Infection rates were up to 50% higher and mean bacterial population densities were 100‐fold higher near the plant base than at the top or in the taproot. 4. Finally, we estimated transmission efficiency of the two leafhoppers when they were confined at either the base or top of inoculated alfalfa plants. Both vectors were inefficient when confined at the top of infected plants and were 20–60% more efficient when confined at the plant base. 5. These results show that vector transmission efficiency is determined by the interaction between leafhopper within‐plant feeding behaviour and pathogen within‐plant distribution. Fine‐scale vector and pathogen overlap is likely to be a requirement generally for efficient transmission of vector‐borne pathogens.     

Detection of Heterobasidion annosum s. l. [(Fr.) Bref.] in Norway Spruce by Polymerase Chain Reaction

Internal transcribed spacer (ITS) sequences of the rDNA repeat unit of Heterobasidion annosum were used to design specific primers for the detection and quantification of this important forest pathogen by polymerase chain reaction (PCR). Specificity of detection was cross‐checked against a variety of other fungi (saprophytes, root pathogens, mycorrhizal fungi) which may occur in the same environment. As little as 1 pg fungal DNA (equiv. to 10–40 genomes) could be detected in 200 ng spruce root DNA (from 1 mg fresh spruce root). The Heterobasidion‐specific primers allowed simultaneous detection of Armillaria spp. in multiplex PCR. The method was successfully applied to increment cores of Norway spruce from the forest region Tharandter Wald (Saxonia, Germany), Oberbärenburg (East Ore Mountains, Saxonia) and Oberschleissheim (north of Munich, Bavaria).     

Landscape‐scale Variation in Pathogen‐suppressive Bacteria in Tropical Dry Forest Soils of Costa Rica

Bacteria in the genus Streptomyces are ubiquitous in soil and are well‐known for their production of diverse secondary metabolites, including antibiotics that can inhibit soil‐borne plant pathogens and suppress disease. Pathogen‐suppressive soil bacteria have the potential to influence plant community composition and diversity, but remain relatively unexplored in tropical forest soils. To estimate the potential for disease suppression among Streptomyces communities in tropical dry forests, we cultured soil‐borne Streptomyces from plots in two forests in northwestern Costa Rica (Santa Rosa and Palo Verde) and quantified antibiotic‐mediated pathogen inhibition against three plant pathogens. The potential for pathogen inhibition and disease suppression by Streptomyces was highly variable across the landscape. Densities of pathogen‐suppressive Streptomyces varied by over ten‐fold and were correlated with soil nutrients across the plots. In particular, Streptomyces communities became more pathogen‐suppressive as labile soil P decreased. Inhibitor densities were significantly higher in Santa Rosa than Palo Verde, which may be related to differences in soil texture and/or plant community composition between the two forests. Our findings suggest potential differences in the degree and specificity of antibiotic‐mediated disease suppression in tropical dry forest soils of Costa Rica, and highlight the need for further studies on the drivers of pathogen‐suppressive phenotypes as well as the consequences of spatially variable pathogen inhibition for plant community composition in tropical forest ecosystems.     

A complex genetic network involving a broad-spectrum locus and strain-specific loci controls resistance to different pathotypes of Aphanomyces euteiches in Medicago truncatula

A higher understanding of genetic and genomic bases of partial resistance in plants and their diversity regarding pathogen variability is required for a more durable management of resistance genetic factors in sustainable cropping systems. In this study, we investigated the diversity of genetic factors involved in partial resistance to Aphanomyces euteiches, a very damaging pathogen on pea and alfalfa, in Medicago truncatula. A mapping population of 178 recombinant inbred lines, from the cross F83005.5 (susceptible) and DZA045.5 (resistant), was used to identify quantitative trait loci for resistance to four A. euteiches reference strains belonging to the four main pathotypes currently known on pea and alfalfa. A major broad-spectrum genomic region, previously named AER1, was localized to a reduced 440 kb interval on chromosome 3 and was involved in complete or partial resistance, depending on the A. euteiches strain. We also identified 21 additive and/or epistatic genomic regions specific to one or two strains, several of them being anchored to the M. truncatula physical map. These results show that, in M. truncatula, a complex network of genetic loci controls partial resistance to different pea and alfalfa pathotypes of A. euteiches, suggesting a diversity of molecular mechanisms underlying partial resistance.     

A high‐resolution genetic map of the cereal crown rot pathogen Fusarium pseudograminearum provides a near‐complete genome assembly

Fusarium pseudograminearum is an important pathogen of wheat and barley, particularly in semi‐arid environments. Previous genome assemblies for this organism were based entirely on short read data and are highly fragmented. In this work, a genetic map of F. pseudograminearum has been constructed for the first time based on a mapping population of 178 individuals. The genetic map, together with long read scaffolding of a short read‐based genome assembly, was used to give a near‐complete assembly of the four F. pseudograminearum chromosomes. Large regions of synteny between F. pseudograminearum and F. graminearum, the related pathogen that is the primary causal agent of cereal head blight disease, were previously proposed in the core conserved genome, but the construction of a genetic map to order and orient contigs is critical to the validation of synteny and the placing of species‐specific regions. Indeed, our comparative analyses of the genomes of these two related pathogens suggest that rearrangements in the F. pseudograminearum genome have occurred in the chromosome ends. One of these rearrangements includes the transposition of an entire gene cluster involved in the detoxification of the benzoxazolinone (BOA) class of plant phytoalexins. This work provides an important genomic and genetic resource for F. pseudograminearum, which is less well characterized than F. graminearum. In addition, this study provides new insights into a better understanding of the sexual reproduction process in F. pseudograminearum, which informs us of the potential of this pathogen to evolve.     

Culture conditions that influence accumulation of zwittermicin A by Bacillus cereus UW85

Bacillus cereus strain UW85 produces an antibiotic, designated zwittermicin A, that is associated with the ability of UW85 to suppress damping-off disease of alfalfa (Medicago sativa) caused by the oomycete pathogen, Phytophthora medicaginis, in a laboratory bioassay. We have identified certain culture conditions that promote or suppress zwittermicin A accumulation by UW85. Maximum accumulation was detected in supernatants of trypticase soy broth cultures after sporulation, which is when cultures of UW85 provide the greatest suppression of damping-off on alfalfa. Inorganic amendments to trypticase soy broth cultures had the following effects on zwittermicin A accumulation and disease suppression: phosphate (50 mM or more) reduced zwittermicin A accumulation and disease suppression; ferric iron (0.25–1.0 mM) enhanced zwittermicin A accumulaiton and disease suppression; micronutrients (manganese, boron, copper, molybdenum, zinc) had no effect on zwittermicin A accumulation or disease suppression. Cultures of UW85 grown in chemically defined minimal medium supplemented with casein hydrolysate or grown in defined medium containing the minimal requirements for growth supplemented with five amino acids (Gln, Arg, Met, Phe, Ile) accumulated zwittermicin A. In minimal medium, alfalfa seed exudate inhibited growth of UW85, whereas alfalfa sprout exudate enhanced zwittermicin A accumulation by 40%. These data indicate that the accumulation of zwittermicin A can be modulated by specific nutrients, inorganic compounds, and plant-derived factors. These results will facilitate the improvement of large-scale purification of zwittermicin A, suggest appropriate conditions under which to conduct further genetic and biochemical analyses, and further substantiate the association between antibiotic accumulation and disease suppression by UW85.     

Biological Control of Phytophthora Root Rot of Pepper Using Trichoderma harzianum and Streptomyces rochei in Combination

A combination of two compatible micro‐organisms, Trichoderma harzianum and Streptomyces rochei, both antagonistic to the pathogen Phytophthora capsici, was used to control root rot in pepper. The population of the pathogen in soil was reduced by 75% as a result. Vegetative growth of the mycelium of P. capsici was inhibited in vitro on the second day after P. capsici and T. harzianum were placed on the opposite sides of the same Petri plate. Trichoderma harzianum was capable of not only arresting the spread of the pathogen from a distance, but also after invading the whole surface of the pathogen colony, sporulating over it. Scanning electron microscopy showed the hyphae of P. capsici surrounded by those of T. harzianum, their subsequent disintegration, and the eventual suppression of the pathogen's growth. Streptomyces rochei produced a zone of inhibition, from which was obtained a compound with antioomycete property secreted by the bacteria. When purified by high‐pressure liquid chromatography, this compound was identified as 1‐propanone, 1‐(4‐chlorophenyl), which seems to be one of the principal compounds involved in the antagonism. A formulation was prepared that maintained the compound's capacity to inhibit growth of the pathogen for up to 2 years when stored at room temperature in the laboratory on a mixture of plantation soil and vermiculite. The two antagonists, added as a compound formulation, were effective at pH from 3.5 to 5.6 at 23–30°C. The optimal dose of the antagonists in the compound formulation was 3.5 × 10⁸ spores/ml of T. harzianum and 1.0 × 10⁹ FCU/ml of S. rochei. This is the first report of a compound biocontrol formulation of these two antagonists with a potential to control root rot caused by P. capsici.