BDP-30, a systemic resistance inducer from Boerhaavia diffusa L., suppresses TMV infection, and displays homology with ribosomeinactivating proteins.

Root extract of Boerhaavia diffusa L. induced systemic resistance in tobacco against Tobacco mosaic virus. A 30 kDa protein was isolated as the active component, called BDP-30 on the basis of the molecular weight and source plant. BDP-30, a glycoprotein, was found to be temperature and protease resistant. It was basic, possessing a pI greater than 9.0. In-gel proteolytic digestion of BDP-30 generated two peptides that possessed the amino acid sequence KLYDIPPLR and KVTLPYSGNYER by LC/MS/MS. Both peptides shared absolute sequence identity with trichosanthin, a ribosome-inactivating protein from Trichosanthes kirilowii, and a 78% and 100% homology respectively with an RIP from Bryonia dioica, bryodin. Further, effort was made to look at the fate of TMV in induced resistant Nicotiana tabacum cv. Xanthi, a systemic host of the virus, at specified days after inoculation in control and treated plants. TMV coat protein (CP) was detected by immunoblot 7 days post inoculation up to 21 days in the control set, but not in treated resistant plants. TMV RNA was detected by RT-PCR using TMV-CP specific primers. Resistant tobacco did not show presence of TMV RNA up to 21 days of inoculation. This suggests that BDP-30 may be suppressing TMV replication.

Interrelationships between Bacillus sp. CHEP5 and Bradyrhizobium sp. SEMIA6144 in the induced systemic resistance against Sclerotium rolfsii and symbiosis on peanut plants.

Plant-growth-promoting bacteria are often used to enhance crop yield and for biological control of phytopathogens. Bacillus sp. CHEP5 is a biocontrol agent that induces systemic resistance (ISR) in Arachis hypogaea L. (peanut) against Sclerotium rolfsii, the causal agent of root and stem wilt. In this work, the effect of the co-inoculation of Bacillus sp. CHEP5 and the peanut nodulating strain Bradyrhizobium sp. SEMIA 6144 was studied on induction of both systemic resistance and nodulation processes. Bradyrhizobium sp. SEMIA 6144 did not affect the ability of Bacillus sp. CHEP5 to protect peanut plants from S. rolfsii by ISR and the priming in challenged-plants, as evidenced by an increment in phenylalanine ammonia-lyase enzyme activity. Additionally, the capacity of Bradyrhizobium sp. SEMIA 6144 to induce nodule formation in pathogen-challenged plants was improved by the presence of Bacillus sp. CHEP5.

LINKAGE MAPPING OF CANDIDATE GENES FOR INDUCE RESISTANCE AND GROWTH PROMOTION BY Trichoderma koningiopsis (Th003) IN TOMATO Solanum lycopersicum / Mapeo de genes candidatos relacionados con inducción de resistencia sistemica y promoción de crecimiento por Trichoderma koningiopsis (Th003) en tomate Solanum lycopersicum

Induced systemic resistance (ISR) is a mechanism by which plants enhance defenses against any stress condition. ISR and growth promotion are enhanced when tomato (Solanum lycopersicum) is inoculated with several strains of Trichoderma ssp. This study aims to genetically map tomato candidate genes involved in ISR and growth promotion induced by the Colombian native isolate Trichoderma koningiopsis Th003. Forty-nine candidate genes previously identified on tomato plants treated with Th003 and T. hamatum T382 strains were evaluated for polymorphisms and 16 of them were integrated on the highly saturated genetic linkage map named “TOMATO EXPEN 2000”. The location of six unigenes was similar to the location of resistance gene analogs (RGAs), defense related ESTs and resistance QTLs previously reported, suggesting new possible candidates for these quantitative trait loci (QTL) regions. The candidate gene-markers may be used for future ISR or growth promotion assisted selection in tomato.

La resistencia sistémica inducida (ISR) es un mecanismo mediante el cual las plantas aumentan sus defensas frente a cualquier condición de estrés. El objetivo de este trabajo fue localizar en el mapa genético de tomate, genes candidatos involucrados en ISR y promoción de crecimiento inducidos por la cepa colombiana nativa Th003 de Trichoderma koningiopsis. Se realizó una búsqueda de polimorfismos en cuarenta y nueve genes candidatos previamente identificados en plantas de tomate inoculadas con Th003 y la cepa T382 de T. hamatum. Diez y seis de estos genes candidatos fueron integrados en el mapa genético de tomate altamente saturado, llamado “TOMATO EXPEN 2000”. La ubicación de seis unigenes fue similar a la localización de genes análogos de resistencia (RGAs), ESTs relacionados con defensa y QTLs de resistencia previamente identificados, sugiriendo posibles nuevos candidatos para estas regiones de QTLs. Los genes candidatos o marcadores pueden ser usados en futuros programas de selección asistida relacionados con ISR o promoción de crecimiento en tomate.

INDUCCIÓN DE RESISTENCIA SISTÉMICA CONTRA Fusarium oxysporum EN TOMATE POR Trichoderma koningiopsis Th003 / Induced Systemic Resistance Against Fusarium oxysporum In Tomato By Trichoderma koningiopsis Th003

Trichoderma koningiopsis Th003 ha mostrado alta eficacia en el control de diferentes fitopatógenos incluyendo Fusarium oxysporum, agente causal de la pudrición del cuello y la raíz del tomate (Solanum lycopersicum Mill.). Con el propósito de estudiar si este agente tiene la capacidad para inducir respuestas sistémicas de defensa, se utilizó como patosistema modelo Fusarium oxysporum -tomate, cuyas plantas se establecieron en cubos de enraizamiento con el sistema radical separado en dos porciones. Cuando Th003 se inoculó en una porción de la raíz 96 h antes de inocular en la otra porción F. oxysporum, se presentó un retraso de la colonización del fitopatógeno en el sistema vascular de la planta, en comparación con las plantas inoculadas solamente con el fitopatógeno. Este resultado sugiere que Th003 estimuló respuestas sistémicas de defensa en la planta, dado que el antagonista y el fitopatógeno permanecieron separados espacialmente. El microorganismo biocontrolador formulado como gránulos dispersables, se aplicó en un cultivo comercial de tomate bajo invernadero y redujo significativamente la incidencia de la pudrición del cuello y las raíces del tomate en 35%, en comparación con el testigo absoluto. El hongo T. koningiopsis Th003 demostró habilidad para controlar F. oxysporum f. sp. radicislycopersici mediante inducción de respuestas de defensa sistémica en las plantas de tomate.

Trichoderma koningiopsis Th003 has proved to be an efficient biocontrol agent of different plant pathogens including Fusarium oxysporum, causing agent of tomato crown and root rot. With the aim to studying whether Th003 has the ability to induce defense systemic responses to control Fusarium oxysporum infection, tomato plants (Solanumlycopersicum Mill.) were sown in pots using split root modified method. When Th003 was applied to one root portion 96 h before inoculating F. oxysporum in the other root portion, delayed colonization of the plant's vascular system was observed as compared with plants inoculated only with the pathogen. Since the antagonist and the pathogen remained spatially separated in the host, the protection effect in plants was attributed to a systemic activity induced by Th003. In a commercial greenhouse the biopesticide based upon Th003 reduced significantly (P<0.05) by 35% the incidence of crown and root rot caused >F. oxysporum f. sp. Lycopersici, compared with untreated control. T. koningiopsis Th003 showed ability to control F. oxysporum f. sp. radicis-lycopersici by inducing systemic defense responses in tomato plants.

Ultrastructures of Colletotrichum orbiculare in the Leaves of Cucumber Plants Expressing Induced Systemic Resistance Mediated by Glomus intraradices BEG110

The colonization of an arbuscular mycorrhizal fungus Glomus intraradices BEG110 in the soil caused a decrease in disease severity in cucumber plants after fungal inoculation with Colletotrichum orbiculare. In order to illustrate the resistance mechanism mediated by G. intraradices BEG110, infection patterns caused by C. orbiculare in the leaves of cucumber plants and the host cellular responses were characterized. These properties were characterized using transmission electron microscopy on the leaves of cucumber plants grown in soil colonized with G. intraradices BEG110. In the untreated plants, inter- and intra-cellular fungal hyphae were observed throughout the leaf tissues during both the biotrophic and necrotrophic phases of infection. The cytoplasm of fungal hyphae appeared intact during the biotrophic phase, suggesting no defense response against the fungus. However, several typical resistance responses were observed in the plants when treated with G. intraradices BEG110 including the formation of sheaths around the intracellular hyphae or a thickening of host cell walls. These observations suggest that the resistance mediated by G. intraradices BEG110 most often occurs in the symplast of the host cells rather than in the apoplast. In addition, this resistance is similar to those mediated by biotic inducers such as plant growth promoting rhizobacteria.

Resistance Induction and Enhanced Tuber Production by Pre-inoculation with Bacterial Strains in Potato Plants against Phytophthora infestans

Efficacy of resistance induction by the bacterial isolates Pseudomonas putida (TRL2-3), Micrococcus luteus (TRK2-2) and Flexibacteraceae bacterium (MRL412), which were isolated from the rhizosphere of plants growing in Jeju Mountain, were tested in a greenhouse. The disease severity caused by Phytophthora infestans was effectively reduced in the potato plants pre-inoculated with bacterial isolates compared with those of the untreated control plants growing in a greenhouse. In order to estimate the level of protection by the bacterial isolates, Mancozeb WP (Diesen M(R), Kyong nong) and DL-3-amino butyric acid (BABA) were pre-treated, whereas Dimethomorph WP (Forum(R), Kyong nong) and phosphonic acid (H3PO3) were post-treated the challenge inoculation with the pathogen. Disease severities of chemical pre-treated as well as post-treated plants were reduced compare to those of the untreated. The disease reduction in the plants pre-treated with Mancozeb WP was the highest, whereas that of post-treated with Dimethomorph WP was the lowest. The yields of plants pre-inoculated with three bacterial isolates were greatly increased than those of control plants. These results suggest that biological control by bacterial isolates might be an alternative strategy against late blight disease in potato plants growing in greenhouse.

Observations of Infection Structures after Inoculation with Colletotrichum orbiculare on the Leaves of Cucumber Plants Pre-inoculated with Two Bacterial Strains Pseudomonas putida or Micrococcus luteus

Infection structures were observed at the penetration sites on the leaves of cucumber plants inoculated with Colletotrichum orbiculare using a fluorescence microscope. The cucumber plants were previously drenched with suspension of bacterial strains Pseudomonas putida or Micrococcus luteus. The plants pre-inoculated with both bacterial strains were resistant against anthracnose after inoculation with C. orbiculare. To investigate the resistance mechanism by both bacterial strains, the surface of infected leaves was observed at the different time after challenge inoculation. At 3 days after inoculation there were no differences in the germination and appressorium formation of conidia of C. orbiculare as well as in the callose formation of the plants between both bacteria pre-inoculated and non-treated. At 5 days, the germination and appressorium formation of the fungal conidia were, however, significantly decreased on the leaves of plants pre-inoculated with M. luteus at the concentration with 1.0 x 10(7) cfu/ml. Furthermore, callose formation of plants cells at the penetration sites was apparently increased. In contrast, there were no defense reactions of the plants at the concentration with 1.0 x 10(6) cfu/ml of M. luteus. Similarly, inoculation P. putida caused no plant resistance at the low concentration, whereas increase of callose formation was observed at the higher concentration. The results of this study suggest that the resistant mechanisms might be differently expressed by the concentration of pre-treatment with bacterial suspension.

Selection and Efficacy of Soil Bacteria Inducing Systemic Resistance Against Colletotrichum orbiculare on Cucumber

Soil bacteria were screened for the ability to control cucumber anthracnose caused by Colletotrichum orbiculare through induced systemic resistance (ISR). Sixty-four bacterial strains having in vitro antifungal activity were used for selecting ISR-inducing strains in cucumber. Cucumber seeds (cv. Baeknokdadagi) were sown in potting mixtures incorporated with the soil bacteria, at a rate of ca. 10(8) cells per gram of the mixture. Two week-old plants were then transplanted into the steam-sterilized soil. Three leaf-stage plants were inoculated with a conidial suspension (5x10(5) conidia/ml) of C. orbiculare. Diseased leaf area (%) and number of lesions per cm2 leaf were evaluated on third leaves of the plants, 5~6 days after inoculation. Among 64 strains tested, nine strains, GC-B19, GC-B35, GK-B18, MM-B22, PK-B14, RC-B41, RC-B64, RC-B65, and RC-B77 significantly (P = 0.05) reduced anthracnose disease compared to the untreated control. In contrast, some bacterial strains promoted susceptibility of cucumber to the disease. From the repeated experiments using the nine bacterial strains, GC-B19, MM-B22, PK-B14, and RC-B65 significantly (P = 0.05) reduced both diseased leaf area (%) and number of lesions per cm2 leaf in at lease one experiment. These strains with control efficacy of 37~80% were determined to be effective ISR-inducing strains.

Different Mechanisms of Induced Systemic Resistance and Systemic Acquired Resistance Against Colletotrichum orbiculare on the Leaves of Cucumber Plants

Defense mechanisms against anthracnose disease caused by Colletotrichum orbiculare on the leaf surface of cucumber plants after pre-treatment with plant growth promoting rhizobacteria(PGPR), amino salicylic acid(ASA) or C. orbiculare were compared using a fluorescence microscope. Induced systemic resistance was mediated by the pre-inoculation in the root system with PGPR strain Bacillus amylolquefaciens EXTN-1 that showed direct antifungal activity to C. gloeosporioides and C. orbiculare. Also, systemic acquired resistance was triggered by the pre-treatments on the bottom leaves with amino salicylic acid or conidial suspension of C. orbiculare. The protection values on the leaves expressing SAR were higher compared to those expressing ISR. After pre-inoculation with PGPR strains no change of the plants was found in phenotype, while necrosis or hypersensitive reaction(HR) was observed on the leaves of plants pre-treated with ASA or the pathogen. After challenge inoculation, inhibition of fungal growth was observed on the leaves expressing both ISR and SAR. HR was frequently observed at the penetration sites of both resistance-expressing leaves. Appressorium formation was dramatically reduced on the leaves of plants pre-treated with ASA, whereas EXTN-1 did not suppress the appressorium formation. ASA also more strongly inhibited the conidial germination than EXTN-1. Conversely, EXTN-1 significantly increased the frequency of callose formation at the penetration sites, but ASA did not. The defense mechanisms induced by C. orbiculare were similar to those by ASA. Based on these results it is suggested that resistance mechanisms on the leaf surface was different between on the cucumber leaves expressing ISR and SAR, resulting in the different protection values.

Systemic Resistance and Expression of the Pathogenesis-Related Genes Mediated by the Plant Growth-Promoting Rhizobacterium Bacillus amyloliquefaciens EXTN-1 Against Anthracnose Disease in Cucumber

Plants have the ability to acquire an enhanced level of resistance to pathogen attack after being exposed to specific biotic stimuli. To obtain plant growth-promoting rhizobacteria inducing resistance against cucumber anthracnose by Colletotrichum orbiculare, more than 800 strains of rhizobacteria were screened in the greenhouse. Among these strains, Bacillus amyloliquefaciens solate EXTN-1 showed significant disease control efficacy on the plants. Induction of pathogenesis-related(PR-la) gene expression by EXTN-1 was assessed using tobacco plants transformed with PR-1a::beta-glucuronidase (GUS) construct. GUS activities of tobacco treated with EXTN-1 and salicylic acid-treated transgenic tobacco were significantly higher than those of tobacco plants with other treatments. Gene expression analyses indicated that EXTN-1 induces the accumulation of defense-related genes of tobacco. The results showed that some defense genes are expressed by the treatment with EXTN-1 suggesting the similar resistance mechanism by salicylic acid.