Systemic Modulation of Gene Expression in Tomato by Trichoderma hamatum 382.

ABSTRACT A light sphagnum peat mix inoculated with Trichoderma hamatum 382 consistently provided a significant (P = 0.05) degree of protection against bacterial spot of tomato and its pathogen Xanthomonas euvesicatoria 110c compared with the control peat mix, even though this biocontrol agent did not colonize aboveground plant parts. To gain insight into the mechanism by which T. hamatum 382 induced resistance in tomato, high-density oligonucleotide microarrays were used to determine its effect on the expression pattern of 15,925 genes in leaves just before they were inoculated with the pathogen. T. hamatum 382 consistently modulated the expression of genes in tomato leaves. We identified 45 genes to be differentially expressed across the replicated treatments, and 41 of these genes could be assigned to at least one of seven functional categories. T. hamatum 382-induced genes have functions associated with biotic or abiotic stress, as well as RNA, DNA, and protein metabolism. Four extensin and extensin-like proteins were induced. However, besides pathogenesis-related protein 5, the main markers of systemic acquired resistance were not significantly induced. This work showed that T. hamatum 382 actively induces systemic changes in plant physiology and disease resistance through systemic modulation of the expression of stress and metabolism genes.

Systemic Resistance Induced by Trichoderma spp.: Interactions Between the Host, the Pathogen, the Biocontrol Agent, and Soil Organic Matter Quality.

ABSTRACT Several factors affect the ability of Trichoderma spp. to provide systemic disease control. This paper focuses on the role of the substrate in which plants are grown, resistance of the host to disease, and the ability of introduced Trichoderma inoculum to spread under commercial conditions. Several reports reveal that foliar disease control provided by Trichoderma spp. is more effective on plants grown in compost-amended media compared with in lower-in-microbial-carrying-capacity sphagnum peat media. In Rhododendron spp., host resistance affects control of Phytophthora dieback provided by Trichoderma spp. For example, T. hamatum 382 (T382) significantly (P = 0.05) suppressed the disease on susceptible cv. Roseum Elegans while plant vigor was increased. The disease was not suppressed, however, on highly susceptible cvs. Aglo and PJM Elite even though the vigor of these plants was increased. Using a strain-specific polymerase chain reaction assay under commercial conditions, it was demonstrated that introduced inoculum of T382 did not spread frequently from inoculated to control compost-amended media. Other Trichoderma isolates typically are abundant in control media within days after potting unless inoculated with a specific Trichoderma isolate. Thus, the low population of isolates that can induce systemic resistance in composting and potting mix environments may explain why most compost-amended substrates do not naturally suppress foliar diseases.

Suppression of Botrytis Blight of Begonia by Trichoderma hamatum 382 in Peat and Compost-Amended Potting Mixes.

Inoculation of an industry standard light sphagnum peat potting mix with Trichoderma hamatum 382 (T382) significantly (P = 0.05) reduced the severity of Botrytis blight, caused by Botrytis cinerea, on begonia plants grown in a greenhouse. In data combined from three experiments, the degree of control provided by T382 did not differ significantly (P = 0.05) from that provided by weekly topical sprays with chlorothalonil. In addition, T382 significantly (P = 0.05) increased shoot dry weight and salability of flowering plants. Incorporation of composted cow manure (5%, vol/vol) into the light peat mix also significantly (P = 0.05) decreased blight severity while shoot dry weight and salability were increased. Blight severity on plants in this compost mix did not differ significantly (P = 0.05) from that on those in the light peat mix inoculated with T382. Finally, T382 and chlorothalonil did not significantly (P = 0.05) affect blight severity, shoot dry weight, or salability of plants grown in the compost mix. Spatial separation was maintained in begonias between the biocontrol agent T382 and the pathogen. It was concluded, therefore, that the decrease in disease severity provided by inoculation of the peat mix with T382 most likely was due to systemic resistance induced in begonia against Botrytis blight. The suppressive effect of the compost mix against Botrytis blight was unusual because composts typically do not provide such effects unless inoculated with a biocontrol agent capable of inducing systemic resistance in plants to disease.

Systemic Resistance Induced by Trichoderma hamatum 382 in Cucumber Against Phytophthora Crown Rot and Leaf Blight.

Phytophthora root rot, crown rot, leaf and stem blight, and fruit rot of cucumber can cause serious losses, and are difficult to control. Although composts can be used successfully for control of Phytophthora root rots, little is known about their effects on Phytophthora diseases of aboveground plant parts. This research shows that the severity of Phytophthora root and crown rot of cucumber caused by Phytophthora capsici was suppressed significantly in cucumber transplants produced in a composted cow manure-amended mix compared with those in a dark sphagnum peat mix. In split root bioassays, Trichoderma hamatum 382 (T382) inoculated into the compost-amended potting mix significantly reduced the severity of Phytophthora root and crown rot on paired roots in the peat mix. This effect did not differ significantly from that provided by a drench with benzothiadiazole (BTH) or mefenoxam (Subdue MAXX). Based on area under disease progress curves, T382 also significantly reduced the severity of Phytophthora leaf blight in transplants produced in the compost mix compared with controls not inoculated with T382. Efficacy of T382 did not differ significantly from that provided by a drench with BTH. T382 re-mained spatially separated from the pathogen in plants in both the split root and leaf blight bioassays, suggesting that these effects were systemic in nature.

Isolation and characterization of rhizobacteria from composts that suppress the severity of bacterial leaf spot of radish.

ABSTRACT Composts can induce systemic resistance in plants to disease. Unfortunately, the degree of resistance induced seems highly variable and the basis for this effect is not understood. In this work, only 1 of 79 potting mixes prepared with different batches of mature, stabilized composts produced from several different types of solid wastes suppressed the severity of bacterial leaf spot of radish caused by Xanthomonas campestris pv. armoraciae compared with disease on plants produced in a nonamended sphagnum peat mix. An additional batch of compost-amended mix that had been inoculated with Trichoderma hamatum 382 (T(382)), which is known to induce systemic resistance in plants, also suppressed the disease. A total of 11 out of 538 rhizobacterial strains isolated from roots of radish seedlings grown in these two compostamended mixes that suppressed bacterial leaf spot were able to significantly suppress the severity of this disease when used as inoculum in the compost-amended mixes. The most effective strains were identified as Bacillus sp. based on partial sequencing of 16S rDNA. These strains were significantly less effective in reducing the severity of this disease than T(382). A combined inoculum consisting of T(382) and the most effective rhizobacterial Bacillus strain was less effective than T(382) alone. A drench applied to the potting mix with the systemic acquired resistance-inducing chemical acibenzolar-S-methyl was significantly more effective than T(382) in several, but not all tests. We conclude that systemic suppression of foliar diseases induced by compost amendments is a rare phenomenon. Furthermore, inoculation of compost-amended potting mixes with biocontrol agents such as T(382) that induce systemic resistance in plants can significantly increase the frequency of systemic disease control obtained with natural compost amendments.

Effect of Compost Amendments on Disease Severity and Yield of Tomato in Conventional and Organic Production Systems.

Field trials were conducted over 2 years to assess the effects of compost amendments on disease development in organic and conventional processing tomato (Lycopersicon esculentum L.) production systems. The incidence of anthracnose fruit rot was reduced in organic tomato plots amended with a high rate of composted cannery wastes compared with the incidence in nonamended control plots in 1998 when disease incidence was high. Marketable yield was increased by 33% in compost-amended organic plots. Plots amended with a high compost rate had more ripe fruit than the nonamended control. The incidence of anthracnose and of total disease on fruit was less on the cultivar OH 8245 than on Peto 696. Total fruit yield of OH 8245 but not Peto 696 in organic plots was increased by amendment with composted cannery wastes. In conventional tomato production, composted yard wastes increased disease severity on foliage both years but reduced bacterial spot incidence on fruit in 1997, when disease pressure was high. The incidence of anthracnose was not affected by composted yard wastes. Marketable and total fruit yields of Peto 696 were not increased in compost-amended conventional plots. The plant activator Actigard reduced foliar disease severity and the incidence of bacterial spot and anthracnose on fruit, while increasing yield of marketable fruit.

Effect of potting mix microbial carrying capacity on biological control of rhizoctonia damping-off of radish and rhizoctonia crown and root rot of poinsettia.

ABSTRACT Potting mixes prepared with dark, highly decomposed Sphagnum peat, with light, less decomposed Sphagnum peat, or with composted pine bark, all three of which were colonized by indigenous microorganisms, failed to consistently suppress Rhizoctonia damping-off of radish or Rhizoctonia crown and root rot of poinsettia. Inoculation of these mixes with Chryseobacterium gleum (C(299)R(2)) and Trichoderma hamatum 382 (T(382)) significantly reduced the severity of both diseases in the composted pine bark mix in which both biocontrol agents maintained high populations over 90 days. These microorganisms were less effective against damping-off in the light and dark peat mixes, respectively, in which populations of C(299)R(2) declined. In contrast, crown and root rot, a disease that is severe late in the crop, was suppressed in all three types of mixes. High populations of T(382) in all three mixes late during the cropping cycle may have contributed to control of this disease.

A rapid bioassay for screening rhizosphere microorganisms for their ability to induce systemic resistance.

ABSTRACT We developed a rapid and miniaturized bioassay for screening large numbers of rhizosphere microorganisms for their ability to induce systemic resistance to bacterial leaf spot of radish caused by Xanthomonas campestris pv. armoraciae. In this bioassay, Pantoea agglomerans strain E278Ar controlled symptoms of disease as effectively as 2,6-dichloroisonicotinic acid when applied to the roots of seedlings produced in growth pouches in a soilless system. E278Ar essentially did not migrate from seedling roots to the foliage. This suggests that induction of systemic resistance could best explain the observed reduction in disease severity. Three mini-Tn5Km-induced mutants of strain E278Ar were isolated that had lost the ability to induce resistance. The bioassay also was used to demonstrate that the fungal biocontrol agent Trichoderma hamatum strain 382 induces systemic resistance in radish. The bioassay required only 14 to 18 days from seeding until rating for disease severity, which is 10 to 14 days less than earlier bioassays.

Precise detection and tracing of Trichoderma hamatum 382 in compost-amended potting mixes by using molecular markers.

Randomly amplified polymorphic DNA (RAPD) analysis and the PCR assay were used in combination with dilution plating on a semiselective medium to detect and enumerate propagules of Trichoderma hamatum 382, a biocontrol agent utilized in compost-amended mixes. Distinct and reproducible fingerprints were obtained upon amplification of purified genomic DNA of T. hamatum 382 with the random primers OPE-16, OPH-19, and OPH-20. Three amplified DNA fragments of 0.35 (OPE-16(0.35)), 0.6 (OPH-19(0.6)), and 0.65 (OPH-20(0.65)) kb were diagnostic for T. hamatum 382, clearly distinguishing it from 53 isolates of four other Trichoderma spp. tested. Some isolates of T. hamatum shared these low-molecular-weight fragments with T. hamatum 382. However, RAPD analysis of isolates of T. hamatum with all three random primers used in consecutive PCR tests distinguished T. hamatum 382 from other isolates of T. hamatum. These three RAPD amplicons were cloned and sequenced, and pairs of oligonucleotide primers for each cloned fragment were designed. Use of the primers in the PCR assay resulted in the amplification of DNA fragments of the same size as the cloned RAPD fragments from genomic DNA of T. hamatum 382. A combination of dilution plating on a semiselective medium for Trichoderma spp. and PCR, with the RAPD primers OPH-19, OPE-16, and OPH-20 or the three sequence-characterized primers, was used successfully to verify the presence of T. hamatum 382 propagules in nine different soil, compost, and potting mix samples. All 23 Trichoderma isolates recovered on semiselective medium from commercial potting mixes fortified with T. hamatum 382 were identified as T. hamatum 382, whereas 274 Trichoderma isolates recovered from the other nine samples were negative in the PCR assay. Thus, this highly specific combination of techniques allowed detection and enumeration of propagules of T. hamatum 382 in fortified compost-amended potting mixes. Sequence-characterized amplified region markers also facilitated the development of a very simple procedure to amplify DNA of T. hamatum 382 directly from fortified compost-amended potting mixes.

Compost and compost water extract-induced systemic acquired resistance in cucumber and Arabidopsis.

ABSTRACT A biocontrol agent-fortified compost mix, suppressive to several diseases caused by soilborne plant pathogens, induced systemic acquired resistance (SAR) in cucumber against anthracnose caused by Colletotrichum orbiculare and in Arabidopsis against bacterial speck caused by Pseudomonas syringae pv. maculicola KD4326. A peat mix conducive to soilborne diseases did not induce SAR. The population size of P. syringae pv. maculicola KD4326 was significantly lower in leaves of Arabidopsis plants grown in the compost mix compared to those grown in the peat mix. Autoclaving destroyed the SAR-inducing effect of the compost mix, and inoculation of the autoclaved mix with nonautoclaved compost mix or Pantoea agglomerans 278A restored the effect, suggesting the SAR-inducing activity of the compost mix was biological in nature. Topical sprays with water extract prepared from the compost mix reduced symptoms of bacterial speck and the population size of pathogenic KD4326 in Arabidopsis grown in the peat mix but not in the compost mix. The peat mix water extract applied as a spray did not control bacterial speck on plants grown in either mix. Topical sprays with salicylic acid (SA) reduced the severity of bacterial speck on plants in the peat mix but did not further reduce the severity of symptoms on plants in the compost mix. The activity of the compost water extract was heat-stable and passed through a 0.2-mum membrane filter. beta-1,3-Glucanase activity was low in cucumber plants grown in either mix, but when infected with C. orbiculare, this activity was induced to significantly higher levels in plants grown in the compost mix than in plants grown in the peat mix. Similar results were obtained for beta-D-glucuronidase (GUS) activity driven by a PR2 (beta-1,3-glucanase) gene promoter in transgenic Arabidopsis plants grown in the compost or peat mix. GUS activity was induced with topical sprays of the compost water extract or SA in plants not inoculated with the pathogen, suggesting that compost-induced disease suppression more than likely involved the potentiation of resistance responses rather than their activation and that compost-induced SAR differed from SAR induced by pathogens, SA, or compost water extract.

Effect of organic matter decomposition level on bacterial species diversity and composition in relationship to pythium damping-off severity.

Rhizosphere bacteria were isolated from root tip segments of cucumber seedlings grown in a suppressive, slightly decomposed light-colored peat mix, a conducive, more decomposed dark-colored peat mix, and a suppressive dark peat mix amended with composted hardwood bark. The bacteria were identified by a gas chromatographic fatty acid methyl ester analysis. The total number of taxa recovered from a single root tip segment ranged from 9 to 18. No single taxon predominated on all root tip segments harvested from any of the mixes. The highest relative population density reached by a given taxon on any root tip segment was 45%. Hill's first and second diversity numbers, the modified Hill's ratio, and Hurlbert's rarefaction method, which were used as measures of species diversity, indicated that the organic matter decomposition level of the potting mixes did not affect bacterial species diversity. Bray-Curtis polar ordination and Dice resemblance functions, however, indicated that the organic matter decomposition level of a mix significantly influenced the composition of bacterial species in the rhizosphere. Pseudomonas spp. and other taxa capable of inducing suppression of pythium damping-off predominated in the suppressive mixes. These organisms were absent from the conducive mix, in which Arthrobacter and Bacillus spp. predominated. Although effective bacterial biocontrol agents were isolated from both the suppressive mixes and the conducive mix, the majority were isolated from the less decomposed suppressive mixes. Finally, the efficacy of strains was significantly greater in the slightly decomposed light peat mix than in the decomposed dark peat mix. Natural disease suppression within these mixes was associated with the organic matter decomposition level and the bacterial species compositions of the mixes.

Characterization of bacteria that suppress rhizoctonia damping-off in bark compost media by analysis of Fatty Acid biomarkers.

Examination of cucumber roots (Cucumis sativus L.) grown in bark compost media and of the surrounding edaphic substrate showed profiles of polar lipid fatty acids commonly found in bacteria. The composition of fatty acids in these profiles differed significantly between roots grown in a medium naturally suppressive to Rhizoctonia damping-off and roots from a conducive medium. Cucumber roots from the suppressive medium had higher proportions of cis-vaccenic acid (18:1 omega 7c) and the iso-branched monoenoic fatty acid i17:1 omega 8 but lower proportions of several iso- and anteiso-branched fatty acids compared with roots from the conducive medium. The concentrations of the bacterial fatty acids were significantly lower in the surrounding media. However, the suppressive and conducive growth substrates had differences in the composition of the bacterial fatty acids similar to those found between the cucumber roots proper. These results suggest major differences in bacterial community composition between suppressive and conducive systems. Fatty acid analyses were also utilized to examine the effects on bacterial community composition of root colonization by Flavobacterium balustinum 299, a biocontrol agent. The concentration of the most prominent fatty acid in this bacterium, i17:1 omega 8, was increased on roots produced from inoculated seeds in a medium rendered suppressive by the treatment. This change was concomitant with a significant increase in the concentration of 18:1 omega 7c, not present in the lipids of the antagonist, indicating a shift in the microflora from a conducive to a suppressive bacterial community.