Survival of the fittest: how the rice microbial community forces Sarocladium oryzae into pathogenicity.

The fungus Sarocladium oryzae (Sawada) causes rice sheath rot and produces the phytotoxins cerulenin and helvolic acid. Both toxins show antimicrobial activity but only helvolic acid production in the rice sheath correlates with virulence. Sarocladium oryzae isolates that differ in their toxin production were used to study their interaction with the rice culturable bacterial endophyte community. The diversity and community structure was defined in the edge of sheath rot lesions, followed by a null model-based co-occurrence analysis to discover pairwise interactions. Non-random pairs were co-cultured to study the nature of the interactions and the role of the toxins herein. Compared to healthy sheaths, endophyte diversity strongly increased when infected with the least virulent S. oryzae isolates producing low amounts of toxins. Virulent S. oryzae isolates did not affect diversity but caused strong shifts in species composition. The endophyte community of healthy rice plants was dominated by B. cereus. This bacterium was enriched in lesions produced by low-virulent S. oryzae isolates and caused hyphal lysis. Contrarily, helvolic acid producers eliminated this bacterium from the sheath endosphere. We conclude that S. oryzae needs to produce antibiotics to defend itself against antagonistic rice endophytes to successfully colonize and infect the rice sheath.

Auxin, Abscisic Acid and Jasmonate Are the Central Players in Rice Sheath Rot Caused by Sarocladium oryzae and Pseudomonas fuscovaginae.

Sheath rot is an emerging rice disease that causes severe yield losses worldwide. The main causal agents are the toxin producers Sarocladium oryzae and Pseudomonas fuscovaginae. The fungus S. oryzae produces helvolic acid and cerulenin and the bacterium P. fuscovaginae produces cyclic lipopeptides. Helvolic acid and the lipopeptide, fuscopeptin, inhibit membrane-bound H+-ATPase pumps in the rice plant. To manage rice sheath rot, a better understanding of the host response and virulence strategies of the pathogens is required. This study investigated the interaction of the sheath rot pathogens with their host and the role of their toxins herein. Japonica rice was inoculated with high- and low-helvolic acid-producing S. oryzae isolates or with P. fuscovaginae wild type and fuscopeptin mutant strains. During infection, cerulenin, helvolic acid and the phytohormones abscisic acid, jasmonate, auxin and salicylic acid were quantified in the sheath. In addition, disease severity and grain yield parameters were assessed. Rice plants responded to high-toxin-producing S. oryzae and P. fuscovaginae strains with an increase in abscisic acid, jasmonate and auxin levels. We conclude that, for both pathogens, toxins play a core role during sheath rot infection. S. oryzae and P. fuscovaginae interact with their host in a similar way. This may explain why both sheath rot pathogens cause very similar symptoms despite their different nature.

How do novel and conventional agri-food wastes, co-products and by-products improve soil functions and soil quality?

Agriculture is estimated to generate about 700 million tons of waste annually in the EU. Novel valorization technologies are developing continuously to recover and recycle valuable compounds and nutrients from waste materials. To close the nutrient loop, low-value agri-food wastes, co-products and by-products (AFWCBs) produced during the valorization process, need to be returned to the soil. However, knowledge on their reaction in soils that is needed to allow efficient and environmentally sound recycling is largely lacking. To this end, we set up a series of laboratory incubation experiments using 10 AFWCBs including insect frass residues made from three different feedstocks, anaerobic digestates from two feedstocks, potato-pulp, rice bran compost, duckweed and two reference crop residues (wheat straw and sugar beet) and measured net N release, C mineralization, dehydrogenase activity (DHA), microbial biomass C (MBC) and community structure. The suppressing potential of frasses and digestates against Rhizoctonia solani was determined using bean. The digestates released the highest net mineral N (50-70%) followed by rice bran compost (55%) and duckweed (30%), while frass made from general food waste and potato-pulp immobilized N like the reference straw for 91 days after incubation. All AFWCBs except digestates significantly increased MBC compared to the control while frasses, potato-pulp and duckweed increased DHA. Frasses and digestates significantly suppressed the development of Rhizoctonia solani in bean plants. AFWCBs from emerging valorizing technologies have the potential to improve microbial activities, C sequestration and may play a significant role in closing the nutrient loop.

Bacillus velezensis as antagonist towards Penicillium roqueforti s.l. in silage: in vitro and in vivo evaluation.

AIMS: The present study was conducted to evaluate the antagonistic effect of Bacillus velezensisNRRL B-23189 towards Penicillium roqueforti s.s. and Penicillium paneum (designated together as P. roqueforti s.l.) in silage conditions. METHODS AND RESULTS: Corn silage conditions were simulated in vitro, and the impact of B. velezensis culture supernatant or cell suspension on P. roqueforti s.l. growth and roquefortine C production was evaluated. The antagonism was promising, but growth of B. velezensis in corn silage infusion was poor. Additionally, an in vivo experiment was carried out with mini-silos containing a mixture of perennial ryegrass and white clover inoculated with P. roqueforti s.l. The applied B. velezensis cell suspension was unsuccessful in reducing P. roqueforti s.l. numbers, but did not compromise the silage acidification. CONCLUSIONS: Although the antagonism observed in vitro was promising, the applied B. velezensis cell suspension could not live up to the expectations in vivo. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, the present study is the first one evaluating the antagonistic properties of B. velezensis towards toxigenic moulds in silage conditions, offering a good base for further research.

Interactions between the oomycete Pythium arrhenomanes and the rice root-knot nematode Meloidogyne graminicola in aerobic Asian rice varieties.

BACKGROUND: Aerobic rice fields are frequently infested by pathogenic oomycetes (Pythium spp.) and the rice root-knot nematode Meloidogyne graminicola. Here, the interaction between Pythium arrhenomanes and Meloidogyne graminicola was studied in rice roots of two aerobic rice varieties. In different experimental set-ups and infection regimes, plant growth, rice yield, Pythium colonization, as well as establishment, development and reproduction of M. graminicola were studied. RESULTS: In this study, it is shown that the presence of P. arrhenomanes delays the establishment, development and reproduction of M. graminicola compared to single nematode infected plants. The delay in establishment and development of M. graminicola becomes stronger with higher P. arrhenomanes infection pressure. CONCLUSIONS: Our data indicate that P. arrhenomanes antagonizes M. graminicola in the rice root and that the plant benefits from this antagonism as shown by the yield data, especially when either of the pathogens is present in high levels.

Does release of encapsulated nutrients have an important role in the efficacy of xylanase in broilers?

The non-starch polysaccharides (NSPs) in cell walls can act as a barrier for digestion of intracellular nutrients. This effect is called "cage effect." Part of the success of fibrolytic enzymes in broiler feed is assumed to be attributed to cage effect reduction. Further, changes in viscosity and potential prebiotic action should also be considered. The aim of this study was to gain insight into the relative importance of the cage effect in xylanase efficacy in broilers. Using a 2×2 factorial design, 24 pens with 30 Ross 308 male chicks were fed corn-soy based diets consisting of normal and freeze-thawed (5 d at -18°C) corn, both with and without xylanase. The freeze-thaw method was used to eliminate the cage effect, whereas a corn-based diet was used to exclude viscosity effects. Body weights (BW), feed intake (FI), and feed conversion ratio (FCR) were determined at d 13, 26, and 39. A balance study was executed at the end of the growing phase. These birds were euthanized at d 34 (non-fasted) to determine the viscosity of digesta, blood metabolites, intestinal morphology, and microbiota composition. During the finisher period, there was a significant interaction between enzyme supplementation and freeze-thawing for FCR, in which FCR was improved by freeze-thawed corn and tended to be improved by normal corn+enzyme compared with the control group. The improvement in performance (finisher period) of freeze-thawed corn and xylanase coincided with increased gut absorption of glucose (based on postprandial plasma concentrations) and increased number of Clostridiumcluster IV in the caecum, and agreed with the higher gut villus height. In addition, xylanase inclusion significantly increased the postprandial plasma glycine and triglycerides concentration, and led to elevated bacterial gene copies of butyryl CoA:acetate CoA-transferase, suggesting a prebiotic effect of xylanase addition through more than just the cage effect reduction. The applied model managed to rule out viscosity by using corn, and it was possible to isolate the cage effect by freeze-thawing the dietary corn.

The endophyte Verticillium Vt305 protects cauliflower against Verticillium wilt.

AIMS: To investigate the interaction between cauliflower and the isolate VerticilliumVt305, obtained from a field suppressive to Verticillium wilt of cauliflower, and to evaluate the ability of VerticilliumVt305 to control Verticillium wilt of cauliflower caused by V. longisporum. METHODS AND RESULTS: Single and combined inoculations of VerticilliumVt305 and V. longisporum were performed on cauliflower seedlings. Symptom development was evaluated, and fungal colonization was measured in the roots, hypocotyl and stem with real-time PCR. No symptoms were observed after single inoculation of VerticilliumVt305, although it colonized the plant tissues. Pre-inoculation of VerticilliumVt305 reduced symptom development and colonization of plant tissues by V. longisporum. CONCLUSIONS: VerticilliumVt305 is an endophyte on cauliflower plants and showed effective biological control of V. longisporum in controlled conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This work can contribute to the development of a sustainable control measure of V. longisporum in Brassicaceae hosts, which is currently not available. Additionally, this study provides evidence for the different roles of Verticillium species present in the agro-ecosystem.

Evaluation of Resistance to Powdery Mildew in Triticale Seedlings and Adult Plants.

Triticale (×Triticosecale) is the intergeneric hybrid between the female parent wheat and the male parent rye. With the expansion of the triticale growing area, powdery mildew emerged on this new host and has become a significant disease on triticale. Recent research demonstrated that this "new" powdery mildew on triticale has emerged through a host range expansion of powdery mildew of wheat. Moreover, this expansion occurred recently and multiple times at different locations in Europe. An effective and environmentally sensitive approach to controlling powdery mildew involves breeding crop plants for resistance. The main goal of this study was to identify the presence of powdery mildew resistance in commercial triticale cultivars. First, the avirulence (AVR) genes and gene complexity carried by this new powdery mildew population on triticale were characterized. Virulence was identified for all the resistance genes evaluated in the present study, and virulence frequencies higher than 50% were recorded on the genes Pm3f, Pm5b, Pm6, Pm7, Pm8, and Pm17. Using molecular markers, the presence of resistance genes Pm3f and Pm17 was identified in certain triticale cultivars. The triticale cultivars were also evaluated for the presence of quantitative resistance at adult plant growth stages in a 2-year field experiment. Despite the high disease pressure, cultivars highly resistant at the adult-plant growth stages were identified. Because 'Grenado' also showed effective race-specific resistance, this cultivar could be of high value for breeding for durable resistance to powdery mildew. Altogether, this study reveals valuable information on the presence of powdery mildew resistance in commercial triticale cultivars, which can be used in breeding programs in triticale. Additionally, this study underscores the need to broaden the base of powdery mildew resistance in triticale through introgression and deployment of new sources of mildew resistance, including quantitative resistance.

qPCR Assays for the Detection of Cylindrocladium buxicola in Plant, Water, and Air Samples.

Cylindrocladium buxicola (syn. C. pseudonaviculatum; teleomorph Calonectria pseudonaviculata) is an important fungal pathogen of Buxus spp. Although widespread in Western Europe, this pathogen has only recently been introduced into North America, where it represents a significant threat to the U.S. and Canadian boxwood industries. Trade of latently infected nursery stock is an important mode of long-distance dissemination and introduction of this pathogen but no methods for detection of latently infected material are available. Also, the pathways for short-distance dispersal of C. buxicola have not been adequately studied. Improved detection methods of this pathogen in air and water samples would benefit future research in this area. We have developed real-time polymerase chain reaction assays for the detection of C. buxicola based on the ribosomal DNA internal transcribed spacer 1 (ITS) and the ß-tubulin 2 gene (TUB). Using a TaqMan probe conjugated with a 3' minor groove binding group (TaqMan MGB probe), the ITS-based assay could reliably detect as little as 10 fg of genomic DNA or 20 copies of cloned target DNA and was approximately 70 times more sensitive than the SYBR Green TUB-based assay. The ITS-based assay provided good but not complete specificity, and is well suited for epidemiological studies. The TUB-based assay, however, proved to be fully specific and can be used for diagnostics. We developed and optimized sample processing and DNA extraction methods for detection of latently present C. buxicola in boxwood plants and quantification of conidia in water and air samples. C. buxicola could be detected in 20 g of plant material, of which only 1 ppm of the tissue was infected, in 10-ml water samples containing as low as 1 conidium/ml, and on Melinex tape pieces representing 12 h of air sampling containing 10 or more conidia. The applicability of the techniques to plant, water, and air samples of practical size was demonstrated.

The expansion of Phytophthora clade 8b: three new species associated with winter grown vegetable crops.

Despite its association with important agricultural crops, Phytophthora clade 8b is a poorly studied group of species. The clade currently consists of three officially described species (Phytophthora porri, P. brassicae and P. primulae) that are host-specific pathogens of leek, cabbages and Primula spp., respectively. However, over the past few decades, several other clade 8b-like Phytophthoras have been found on a variety of different host plants that were all grown at low temperatures in winter seasons. In this study, a collection of 30 of these isolates was subjected to a phylogenetic study using two loci (the rDNA ITS region and the mitochondrial cox1 gene). This analysis revealed a clear clustering of isolates according to their host plants. To verify whether these isolates belong to separate species, a detailed morphological study was conducted. On the basis of genetic and morphological differences and host specificity, we now present the official description of three new species in clade 8b: Phytophthora cichorii sp. nov., P. dauci sp. nov. and P. lactucae sp. nov. Two other groups of isolates (Phytophthora taxon castitis and Phytophthora taxon parsley) might also represent new species but the data available at this time are insufficient for an official description. This brings Phytophthora clade 8b to a group of six species that are all host-specific, slow-growing and specifically infect herbaceous crops at low temperatures.

Overexpression of arginase in Arabidopsis thaliana influences defence responses against Botrytis cinerea.

Arabidopsis possesses two arginase-encoding genes, ARGAH1 and ARGAH2, catalysing the catabolism of arginine into ornithine and urea. Arginine and ornithine are both precursors for polyamine biosynthetic pathways. We observed an accumulation of ARGAH2 mRNA in Arabidopsis upon inoculation with the necrotrophic pathogen Botrytis cinerea. Transgenic lines displaying either overexpression of ARGAH2 or simultaneous silencing of both Arabidopsis arginase-encoding genes were created and their resistance to B. cinerea infection evaluated. Overexpression of arginase resulted in changes in amino acid accumulation, while polyamine levels remained largely unaffected. Silencing lines were affected in both amino acid and putrescine accumulation. Arabidopsis plants overexpressing the arginase gene were less susceptible to B. cinerea, whereas silencing lines remained as susceptible as the wild type. We discuss how arginase might interact with plant defence mechanisms. These results provide new insights into amino acid metabolic changes under stress.

Comparing systemic defence-related gene expression changes upon migratory and sedentary nematode attack in rice.

Complex defence signalling pathways, controlled by different hormones, are known to be involved in the reaction of plants to a wide range of biotic and abiotic stress factors. Here, we studied the differential expression of genes involved in stress and defence responses in systemic tissue of rice infected with the root knot nematode (RKN) Meloidogyne graminicola and the migratory root rot nematode Hirschmanniella oryzae, two agronomically important rice pathogens with very different lifestyles. qRT-PCR revealed that all investigated systemic tissues had significantly lower expression of isochorismate synthase, a key enzyme for salicylic acid production involved in basal defence and systemic acquired resistance. The systemic defence response upon migratory nematode infection was remarkably similar to fungal rice blast infection. Almost all investigated defence-related genes were up-regulated in rice shoots 3 days after root rot nematode attack, including the phenylpropanoid pathway, ethylene pathway and PR genes, but many of which were suppressed at 7 dpi. Systemic shoot tissue of RKN-infected plants showed similar attenuation of expression of almost all studied genes already at 3 dpi, with clear attenuation of the ethylene pathway and methyl jasmonate biosynthesis. These results provide an interesting starting point for further studies to elucidate how nematodes are able to suppress systemic plant defence mechanisms and the effect in multitrophic interactions.

Detection of Multiple Verticillium Species in Soil Using Density Flotation and Real-Time Polymerase Chain Reaction.

Wet sieving of soil samples, followed by plating on semi-selective medium and microscopic analysis, is the most commonly used technique to quantify microsclerotia-forming Verticillium species in soil. However, the method is restricted to small samples, does not allow easy differentiation between species, and takes several weeks to complete. This study describes an alternative method to test 100-g soil samples for three Verticillium species (V. tricorpus, V. dahliae, and V. longisporum) using density flotation-based extraction of microsclerotia followed by new real-time polymerase chain reaction (PCR) assays. Primers for these real-time PCR assays were designed to the ribosomal DNA internal transcribed spacer for V. tricorpus and the ß-tubulin gene for V. dahliae + V. longisporum and V. longisporum. Tests with artificially and naturally infested soils showed that the new method is reproducible and sensitive (0.1 to 0.5 microsclerotia/g soil), allows differentiation among the three species, and can be completed in one day. The results of the new method and the wet-sieving method were highly correlated for V. tricorpus (R2 = 0.78), but not for V. dahliae/V. longisporum, probably due to the loss of germinability of V. dahliae/V. longisporum microsclerotia during prolonged dry storage of the soil.

N-Acylhomoserine lactone quorum-sensing signalling in antagonistic phenazine-producing Pseudomonas isolates from the red cocoyam rhizosphere.

Forty fluorescent Pseudomonas strains isolated from white and red cocoyam roots were tested for their ability to synthesize N-acyl-l-homoserine lactones (acyl-HSLs). Remarkably, only isolates from the red cocoyam rhizosphere that were antagonistic against the cocoyam root rot pathogen Pythium myriotylum and synthesized phenazine antibiotics produced acyl-HSLs. This supports the assumption that acyl-HSL production is related to the antagonistic activity of the strains. After detection, the signal molecules were identified through TLC-overlay and liquid chromatography-multiple MS (LC-MS/MS) analysis. In our representative strain, Pseudomonas CMR12a, production of the signal molecules could be assigned to two quorum-sensing (QS) systems. The first one is the QS system for phenazine production, PhzI/PhzR, which seemed to be well conserved, since it was genetically organized in the same way as in the well-described phenazine-producing Pseudomonas strains Pseudomonas fluorescens 2-79, Pseudomonas chlororaphis PCL1391 and Pseudomonas aureofaciens 30-84. The newly characterized genes cmrI and cmrR make up the second QS system of CMR12a, under the control of the uncommon N-3-hydroxy-dodecanoyl-homoserine lactone (3-OH-C12-HSL) and with low similarity to other Pseudomonas QS systems. No clear function could yet be assigned to the CmrI/CmrR system, although it contributes to the biocontrol capability of CMR12a. Both the PhzI/PhzR and CmrI/CmrR systems are controlled by the GacS/GacA two-component regulatory system.

Biosurfactants are involved in the biological control of Verticillium microsclerotia by Pseudomonas spp.

AIMS: To examine the effect of previously described bacterial antagonists on the viability of Verticillium microsclerotia in vitro and to elucidate the possible modes of action of bacterial strains in the suppression of Verticillium microsclerotia viability. METHODS AND RESULTS: A microplate assay was developed to test the suppressive effect of well-defined Pseudomonas spp. on the viability of Verticillium microsclerotia in vitro. Experiments using phenazine- and biosurfactant-deficient mutants indicated that biosurfactants and phenazine-1-carboxylic acid play a role in the suppression of microsclerotia viability by Pseudomonas spp. In addition, microsclerotia colonization tests revealed that Pseudomonas spp. are able to colonize the surface of the microsclerotia, but not the inner matrix. Growth response curves showed that the population levels of Pseudomonas spp. increased when they were in the vicinity of Verticillium microsclerotia, indicating that Pseudomonas spp. may utilize nutrients from the microsclerotia for their growth. CONCLUSIONS: Pseudomonas spp. seem to be good candidates for Verticilllium microsclerotia biocontrol. Biosurfactant production is one of the main mechanisms involved in their mode of action. SIGNIFICANCE AND IMPACT OF THE STUDY: This line of work may contribute to a better understanding of biological control agents and their working mechanisms.

Characterization of CMR5c and CMR12a, novel fluorescent Pseudomonas strains from the cocoyam rhizosphere with biocontrol activity.

AIM: To screen for novel antagonistic Pseudomonas strains producing both phenazines and biosurfactants that are as effective as Pseudomonas aeruginosa PNA1 in the biocontrol of cocoyam root rot caused by Pythium myriotylum. MATERIAL AND RESULTS: Forty pseudomonads were isolated from the rhizosphere of healthy white and red cocoyam plants appearing in natural, heavily infested fields in Cameroon. In vitro tests demonstrated that Py. myriotylum antagonists could be retrieved from the red cocoyam rhizosphere. Except for one isolate, all antagonistic isolates produced phenazines. Results from whole-cell protein profiling showed that the antagonistic isolates are different from other isolated pseudomonads, while BOX-PCR revealed high genomic similarity among them. 16S rDNA sequencing of two representative strains within this group of antagonists confirmed their relatively low similarity with validly described Pseudomonas species. These antagonists are thus provisionally labelled as unidentified Pseudomonas strains. Among the antagonists, Pseudomonas CMR5c and CMR12a were selected because of their combined production of phenazines and biosurfactants. For strain CMR5c also, production of pyrrolnitrin and pyoluteorin was demonstrated. Both CMR5c and CMR12a showed excellent in vivo biocontrol activity against Py. myriotylum to a similar level as Ps. aeruginosa PNA1. CONCLUSION: Pseudomonas CMR5c and CMR12a were identified as novel and promising biocontrol agents of Py. myriotylum on cocoyam, producing an arsenal of antagonistic metabolites. SIGNIFICANCE AND IMPACT OF THE STUDY: Present study reports the identification of two newly isolated fluorescent Pseudomonas strains that can replace the opportunistic human pathogen Ps. aeruginosa PNA1 in the biocontrol of cocoyam root rot and could be taken into account for the suppression of many plant pathogens.

Molecular detection of Puccinia horiana Henn. the causal agent of Chrysanthemum white rust.

Chrysanthemum white rust is one of the most important foliar diseases of pot chrysanthemum and is a quarantine pathogen in many countries. Under conducive environmental conditions, it has the potential to completely destroy susceptible cultivars. This is mainly avoided through frequent preventive fungicide applications. As part of a research program to develop a disease warning system, a molecular detection method was developed. To determine the nucleotide sequence of the nuclear rDNA-ITS (internal transcribed spacer) region of P. horiana, 56 isolates were collected between 2003 and 2006 from diseased commercial chrysanthemum plants from different national and international geographical areas. DNA was isolated from the basidiospores or teliospores from several isolates and the rDNA-ITS region was cloned and sequenced. Based on the limited variability in rDNA-ITS sequence between these isolates, several primer pairs were designed and tested for detection through conventional and real-time PCR. Specificity of detection was cross-checked against a variety of other fungi (saprophytes and other rusts) that may occur in the same environment, and against DNA of healthy chrysanthemum leaves. Using the best primers, the PCR-based methods successfully detected all the P. horiana isolates tested, while no signal was observed with other rust species up to 1 ng non target genomic DNA template. The limit of detection of P. horiana DNA in conventional, nested and real-time PCR was 10 pg, 10 fg and 10 fg, respectively. The DNA extraction method and PCR template concentration were optimized to maximize the recoverability of the pathogen from infected plant tissue. Using the optimized real-time PCR method, the pathogen could be detected in washed plant tissue, 9 hours after inoculation. Hence, this method allows detection of the P. horiana in any part of its latent stage and will also serve as a tool for studying the biology and epidemiology of the pathogen.

Pseudomonads associated with midrib rot and soft rot of butterhead lettuce and endive.

During the past ten years, bacterial soft rot and midrib rot of glasshouse-grown butterhead lettuce (Lactuca sativa L. var. capitata) and field-grown endive (Cichorium endivia L.) has become increasingly common in the region of Flanders, Belgium. Severe losses and reduced market quality caused by bacterial rot represent an important economical threat for the production sector. Symptoms of midrib rot are a brownish rot along the midrib of one or more inner leaves, often accompanied by soft rot of the leaf blade. Twenty-five symptomatic lettuce and endive samples were collected from commercial growers at different locations in Flanders. Isolations of dominant bacterial colony types on dilution plates from macerated diseased tissue extracts yielded 282 isolates. All isolates were characterized by colony morphology and fluorescence on pseudomonas agar F medium, oxidase reaction, and soft rot ability on detached chicory leaves. Whole-cell fatty acid methyl esters profile analyses identified the majority of isolates (85%) as belonging to the Gammaproteobacteria, which included members of the family Enterobacteriaceae (14%) and of the genera Pseudomonas (73%), Stenotrophomonas (9%), and Acinetobacter (3%). Predominant bacteria were a diverse group of fluorescent Pseudomonas species. They were further differentiated based on the non-host hypersensitive reaction on tobacco and the ability to rot potato slices into 4 phenotypic groups: HR-/P- (57 isolates), HR-/P+ (54 isolates), HR+/P (16 isolates) and HR+/P+ (35 isolates). Artificial inoculation of suspensions of HR-, pectolytic fluorescent pseudomonads in the leaf midrib of lettuce plants produced various symptoms of soft rot, but they did not readily cause symptoms upon spray inoculation. Fluorescent pseudomonads with phenotype HR+ were consistently isolated from typical dark midrib rot symptoms, and selected isolates reproduced the typical midrib rot symptoms when spray-inoculated onto healthy lettuce plants.