Induced Systemic Resistance and Promotion of Plant Growth by Bacillus spp

ABSTRACT Elicitation of induced systemic resistance (ISR) by plant-associated bacteria was initially demonstrated using Pseudomonas spp. and other gram-negative bacteria. Several reviews have summarized various aspects of the large volume of literature on Pseudomonas spp. as elicitors of ISR. Fewer published accounts of ISR by Bacillus spp. are available, and we review this literature for the first time. Published results are summarized showing that specific strains of the species B. amyloliquefaciens, B. subtilis, B. pasteurii, B. cereus, B. pumilus, B. mycoides, and B. sphaericus elicit significant reductions in the incidence or severity of various diseases on a diversity of hosts. Elicitation of ISR by these strains has been demonstrated in greenhouse or field trials on tomato, bell pepper, muskmelon, watermelon, sugar beet, tobacco, Arabidopsis sp., cucumber, loblolly pine, and two tropical crops (long cayenne pepper and green kuang futsoi). Protection resulting from ISR elicited by Bacillus spp. has been reported against leaf-spotting fungal and bacterial pathogens, systemic viruses, a crown-rotting fungal pathogen, root-knot nematodes, and a stem-blight fungal pathogen as well as damping-off, blue mold, and late blight diseases. Reductions in populations of three insect vectors have also been noted in the field: striped and spotted cucumber beetles that transmit cucurbit wilt disease and the silver leaf whitefly that transmits Tomato mottle virus. In most cases, Bacillus spp. that elicit ISR also elicit plant growth promotion. Studies on mechanisms indicate that elicitation of ISR by Bacillus spp. is associated with ultrastructural changes in plants during pathogen attack and with cytochemical alterations. Investigations into the signal transduction pathways of elicited plants suggest that Bacillus spp. activate some of the same pathways as Pseudomonas spp. and some additional pathways. For example, ISR elicited by several strains of Bacillus spp. is independent of salicylic acid but dependent on jasmonic acid, ethylene, and the regulatory gene NPR1-results that are in agreement with the model for ISR elicited by Pseudomonas spp. However, in other cases, ISR elicited by Bacillus spp. is dependent on salicylic acid and independent of jasmonic acid and NPR1. In addition, while ISR by Pseudomonas spp. does not lead to accumulation of the defense gene PR1 in plants, in some cases, ISR by Bacillus spp. does. Based on the strains and results summarized in this review, two products for commercial agriculture have been developed, one aimed mainly at plant growth promotion for transplanted vegetables and one, which has received registration from the U.S. Environmental Protection Agency, for disease protection on soybean.     

Evaluation of Trichoderma spp. as a Biocontrol Agent Against Soilborne Fungi and Plant-parasitic Nematodes in Israel

The optimal conditions required to market Trichoderma as a biocontrol agent against soilborne fungi and nematodes are discussed. These include a proper formulation, an efficient delivery system, and alternative methods for Trichoderma's application.The implementation of Trichoderma in integrated pest management (IPM) can be achieved using a soil treatment which combines reduced amounts of biocides/fungicides and the Trichoderma preparation. Biocontrol activity can be increased by combining two (or more) types of biocontrol agents. Moreover, the construction of a genetically modified Trichoderma can lead to the improvement of certain traits which are absent or not highly expressed in the native microorganism isolated from its natural habitat.Different Trichoderma harzianum and T. lignorum isolates were tested for their nematicidal activity against the root-knot nematode Meloidogyne javanica. In short-term experiments, improved growth of nematode-infected plants and decreases in the root-galling index and the number of eggs per gram of root were achieved when nematode-infested soils were pre- exposed to the T. harzianum preparations. A long-term experiment resulted in improved growth and higher yield of nematode-infected plants, but no significant change in the galling index, either by pre-exposure of the fungus to the soil or by enrichment in the root-ball.As biocontrol is an integral part of the IPM philosophy, judicious use of Trichoderma against soilborne pathogens, when demonstrated to be consistently effective, practical and economic, can serve as a model for the introduction and implementation of other biocontrol means into IPM.     

The Molecular Biology of the Interactions Between Trichoderma spp., Phytopathogenic Fungi, and Plants

ABSTRACT Trichoderma-based biofungicides are a reality in agriculture, with more than 50 formulations today available as registered products worldwide. Several strategies have been applied to identify the main genes and compounds involved in this complex, three-way cross-talk between the fungal antagonist, the plant, and microbial pathogens. Proteome and genome analysis have greatly enhanced our ability to conduct holistic and genome-based functional studies. We have identified and determined the role of a variety of novel genes and gene-products, including ABC transporters, enzymes and other proteins that produce or act as novel elicitors of induced resistance, proteins responsible for a gene-for-gene avirulent interaction between Trichoderma spp. and plants, mycoparasitism-related inducers, plant proteins specifically induced by Trichoderma, etc. We have transgenically demonstrated the ability of Trichoderma spp. to transfer heterologous proteins into plant during root colonization, and have used green fluorescent protein and other markers to study the interaction in vivo and in situ between Trichoderma spp. and the fungal pathogen or the plant.     

Rhizobacteria-mediated Induced Systemic Resistance: Triggering, Signalling and Expression

Selected strains of rhizosphere bacteria reduce disease by activating a resistance mechanism in the plant named rhizobacteria-mediated induced systemic resistance (ISR). Rhizobacteria-mediated ISR resembles pathogen-induced systemic acquired resistance (SAR) in that both types of induced resistance render uninfected plant parts more resistant towards a broad spectrum of plant pathogens. Some rhizobacteria trigger the salicylic acid (SA)-dependent SAR pathway by producing SA at the root surface. In other cases, rhizobacteria trigger a different signalling pathway that does not require SA. The existence of a SA-independent ISR pathway has been demonstrated in Arabidopsis thaliana. In contrast to pathogen-induced SAR, ISR induced by Pseudomonas fluorescens WCS417r is independent of SA accumulation and pathogenesis-related (PR) gene activation but, instead, requires responsiveness to the plant hormones jasmonic acid (JA) and ethylene. Mutant analyses showed that ISR follows a novel signalling pathway in which components from the JA and ethylene response are successively engaged to trigger a defensive state that, like SAR, is controlled by the regulatory factor NPR1. Interestingly, simultaneous activation of both the JA/ethylene-dependent ISR pathway and the SA-dependent SAR pathway results in an enhanced level of protection. Thus combining both types of induced resistance provides an attractive tool for the improvement of disease control. This review focuses on the current status of our research on triggering, signalling, and expression of rhizobacteria-mediated ISR in Arabidopsis.     

Role of Salicylic Acid in Systemic Resistance Induced by Pseudomonas spp. Against Pythium aphanidermatum in Cucumber Roots

Pseudomonas corrugata strain 13 and P. aureofaciens strain 63-28, applied to roots, induced systemic resistance against Pythium aphanidermatum in cucumber roots. Salicylic acid (SA) from bacterial culture or plant tissues was quantified by high performance liquid chromatography. Both strains produced SA in King's B broth and also induced cucumber root to accumulate endogenous SA one day after bacterial inoculation. Using a split root system, more SA accumulated in roots treated with bacteria than in distant roots on the opposite side of the root system in the first two days, but this difference disappeared after 3–4 days. SA levels were significantly higher in plants treated with bacteria compared to the split control, from one to five days after bacterization. SA did not inhibit mycelial growth of Pythium aphanidermatum at 100–200µgml^–1 in vitro, but higher levels inhibited mycelial growth. Zoospore germination increased at concentrations of 10–500µgml^–1, but decreased at 1000µgml^–1 compared to lower concentrations. Exogenously applied SA failed to induce local or systemic resistance against a challenge infection by the pathogen in planta. The results of this study show that exogenous applied SA does not induce systemic resistance to cucumber root rot caused by P. aphanidermatum, but endogenous SA accumulation in cucumber roots may be involved in induced systemic resistance.     

Induced Systemic Resistance (ISR) by Pseudomonas spp. Impairs Pre- and Post-Infection Development of Pythium aphanidermatum on Cucumber Roots

The effect of induced systemic resistance (ISR) by Pseudomonas rhizobacteria on the pre- and post-infection development of Pythium aphanidermatum on cucumber roots was investigated. Cucumber plants (cv. Corona) were grown in vermiculite, roots were split with one side bacterized with Pseudomonas corrugata strain 13 or P. aureofaciens strain 63-28 (bacterized roots) and the other distant side was treated with water (distant, induced roots). For the non-induced control, roots on the bacterized side were treated with buffer instead of the bacterial treatment. Intact, non-split roots were also treated with the bacteria or buffer as a control. Cucumber root tissue from these treatments were harvested and incubated with a zoospore suspension of P. aphanidermatum for three hours. Most of the zoospores in the suspension were stimulated to encyst or germinate. The numbers of germinated zoospores were significantly decreased on distant induced cucumber roots in comparison to non-induced controls. Germination was also reduced on intact bacterized roots, compared to controls. There was less attachment, germ tube production and penetration on roots bacterized or induced by the rhizobacteria compared to non-induced roots. Effects were significantly greater on bacterized roots (roots colonized by bacteria) compared to distant induced roots (roots with the opposite side bacterized). Systemic resistance induced by the two Pseudomonas spp. also reduced pathogen spread on split cucumber roots in planta. Crown infection from induced or bacterized roots was delayed for four to six days in comparison to the non-induced control. Results indicated that Pseudomonas spp. can exert both an indirect influence on P. aphanidermatum zoospore behaviour and infection via induced systemic resistance (ISR) and a local influence via antibiosis or local induced resistance.     

Substrate Colonization, Strain Competition, Enzyme Production In Vitro, and Biocontrol of Pythium ultimum by Trichoderma spp. Isolates P1 and T3

The antagonistic Trichoderma spp. isolates P1 and T3 differed in their ability to colonize and to compete in sphagnum peat moss and on wood chips. In peat supplemented with straw, isolate T3 produced twice as many colony forming units (cfu) as isolate P1. On wood chips, the two isolates formed a similar number of cfu. When the two Trichoderma isolates were cultivated together approximately 85–90% of the cfu were from T3 on both substrates. The presence of Pythium ultimum in peat amended with straw did not influence the number of Trichoderma cfu formed. The two Trichoderma isolates produced different amounts of hydrolytic enzymes both in liquid cultures and in peat. Seven different enzyme activities were tested. Enzyme production by T. harzianum isolate T3 was less influenced by the type of carbon source amendment than that of isolate T. atroviride P1. Culture filtrates of isolate P1 grown on complex carbon sources were high in endochitinase activity, whereas cellulase and endo-1,3--glucanase activities were more pronounced in filtrates of isolate T3. There was no significant difference between the two isolates in their ability to protect cucumber seedlings against P. ultimum while the combination of the two fungi resulted in significantly less biocontrol than each isolate alone.     

Evidence for Antibiosis and Induced Host Defense Reactions in the Interaction Between Verticillium lecanii and Penicillium digitatum, the Causal Agent of Green Mold

ABSTRACT Chronological events of the intercellular interaction between Verticillium lecanii and the postharvest pathogen Penicillium digitatum were investigated by transmission electron microscopy and gold cytochemistry. Growth inhibition of P. oligandrum as a response to V. lecanii attack correlated with striking host changes including retraction of the plasma membrane and cytoplasm disorganization. Such changes were associated with the deposition on the inner host cell surface of a chitin- and cellulose-enriched material which appeared to be laid down as a structural defense reaction. The accumulation of chitin in the newly formed material correlated with a decrease in the amount of wallbound chitin. However, the deposition of cellulose appeared to correspond to a de novo synthesis, as evidenced by the occurrence of cellulose-containing vesicles which released their content in the space between the invaginated plasma membrane and the host cell wall. Results of the present study provide the first ultrastructural and cytochemical evidence that antagonism, triggered by V. lecanii, is a multifaceted process in which antibiosis, with alteration of the host hyphae prior to contact with the antagonist, appears to be the key process in the antagonism against P. digitatum.     

Local and Systemic Induced Resistance to the Root-Knot Nematode in Tomato by DL-beta-Amino-n-Butyric Acid

ABSTRACT Chemical inducers of pathogenesis-related proteins and plant resistance were applied to tomato plants, with the aim of inducing resistance to the root-knot nematode Meloidogyne javanica. Relative to control plants, foliar spray and soil-drenching with dl-beta-amino-n-butyric acid (BABA) reduced root-galling 7 days after inoculation, as well as the number of eggs 30 days after inoculation. Other chemicals (alpha- and gamma-amino-n-butyric acid, jasmonic acid, methyl jasmonate, and salicylic acid) were either phytotoxic to tomato plants or did not improve control of root-knot nematodes. Fewer second-stage juveniles invaded BABA-treated tomato roots, and root-galling indices were lower than in control tomato plants. Resistance phenomena in seedlings lasted at least 5 days after spraying with BABA. Nematodes invading the roots of BABA-treated seedlings induced small, vacuolate giant cells. Postinfection treatment of tomato plants with BABA inhibited nematode development. It is speculated that after BABA application tomato roots become less attractive to root-knot nematodes, physically harder to invade, or some substance(s) inhibiting nematode or nematode feeding-site development is produced in roots.     

无致病性腐霉的生防作用和诱导防卫反应

寡雄腐霉Pythium oligandrum作为一种重要的土传病害生防因子越来越受到关注,它能在多种重要农作物根围定殖,对20多种病原真菌或其它卵菌具有拮抗或寄生作用,并能诱导植物产生防卫反应.寡雄腐霉产生的拟激发素寡雄蛋白诱导番茄系统获得抗性,抵抗寄生疫霉Phytophthora parasitica和尖孢镰刀菌羽扇豆-番茄枯萎病菌Fusarium oxysporum f.sp.radicis-lycopersici侵入,显著降低病害的发生率.作者对寡雄腐霉的分离与鉴定、寄主种类、寄生作用、抗生作用和拟激发素寡雄蛋白诱导番茄系统获得抗性等方面的研究进行了综述.     

哈茨木霉NF9菌株对水稻的诱导抗病性

在温室条件下用哈茨木霉NF9菌株进行种子处理，诱导水稻感病品种原丰早幼苗对稻-瘟病和白叶枯病的抗病性，其抗性水平为中抗，对稻瘟病菌不同菌株(SYl、H16)以及对白叶枯病菌不同菌株(CRl、CR7)的抗性表现无显著差异。经木霉种子处理的水稻幼苗接种稻瘟病菌或白叶枯病菌后，过氧化物酶及苯丙氨酸转氨酶活性增强，在某一时段显著高于未经木霉种子处理的对照。诱导抗性可能是哈茨木霉NF9菌株的生防机制之一。     

Interactions Among a Soil Organic Amendment, Nematodes, and the Nematode-Trapping Fungus Dactylellina candidum

ABSTRACT When alfalfa leaves (Medicago sativa) are added to soil, both the nematode-trapping fungus Dactylellina candidum and microbivorous nematodes increase. To determine whether the response of the fungus to alfalfa depends on consumption of bacterivorous and fungivorous nematodes, soil microcosm experiments were performed. D. candidum did not increase if alfalfa leaves were added to soil lacking nematodes, but did increase if nematodes were added to soil lacking alfalfa leaves. Although these results indicate that the response of D. candidum to alfalfa depends on nematodes, D. candidum and microbivorous nematodes did not exhibit classical predator-prey dynamics (i.e., D. candidum seldom reduced numbers of resident nematodes and, after initially increasing in alfalfa amended soil, numbers of D. candidum then decreased, whereas numbers of nematodes continued to increase). Fungivorous nematodes were abun dant in alfalfa-amended soil, and their potential to suppress trapping fungi requires more research.     

Short-term Cover Crop Decomposition in Organic and Conventional Soils: Characterization of Soil C, N, Microbial and Plant Pathogen Dynamics

Stages of oat–vetch cover crop decomposition were characterized over time in terms of carbon and nitrogen cycling, microbial activity and damping-off pathogen dynamics in organically and conventionally managed soils in a field and a controlled incubation experiment. A measurement of relative growth consisting of radial growth of a fungal colony over non-sterilized soil divided by that over sterilized soil was used as an assay of suppressiveness. No differences in relative growth of Pythium aphanidermatum and Rhizoctonia solani were detected between organic and conventionally managed soils amended with cover crop residue. Significant effects of cover crop decomposition stage on the relative growth of both pathogens were obtained. Relative growth of P. aphanidermatum was highest just after incorporation and decreased 3 weeks after incorporation. Relative growth of R. solani was highest about 20 days after incorporation, and decreased 2 weeks later in the organic system, but continued to increase in the conventional system. In both experiments, the N or C content, C:N ratio or dry weight of retrieved debris were significantly correlated with relative growth of P. aphanidermatum. Relative growth of R. solani was significantly correlated with the C:N ratio of soil or the C or N content of debris. Microbial activity was not consistently associated with relative growth of either pathogen.     

Different Patterns of Host Genes are Induced in Rice by Pseudomonas syringae, a Biological Inducer of Resistance, and the Chemical Inducer Benzothiadiazole (BTH)

Rice seedlings treated with the synthetic compound benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH) acquired resistance to subsequent attack by the rice blast fungus Magnaporthe grisea (Hebert) Barr. BTH (trade name Bion) has been released to the market as a plant protecting agent for rice. Here, we analysed the pattern of expressed genes in rice plants treated with BTH, and compared this pattern with those induced by the formerly discovered resistance inducer 2,6-dichloroisonicotinic acid (INA) and by Pseudomonas syringae pv. syringae, a non-host pathogen inducing a hypersensitive response. Both INA and BTH induced similar patterns of genes, suggesting that these compounds are functional analogues. In contrast, the patterns induced by the chemical inducers and by P. syringae were clearly dissimilar.     

Induced Resistance by Penicillium oxalicum Against Fusarium oxysporum f. sp. lycopersici: Histological Studies of Infected and Induced Tomato Stems

ABSTRACT Tomato (Lycopersicon esculentum) plants of 'Lorena' were induced with a conidial suspension (10(7) conidia per ml) of Penicillium oxalicum before inoculation with Fusarium oxysporum f. sp. lycopersici, the wilt pathogen. Histological changes occurred in plants under both growth chamber and glasshouse culture conditions and there was a reduction of disease severity. In noninduced plants, the pathogen produced almost a complete loss of cambium (75 to 100% reduction), an increase in the number of bundles, and a decrease in the number of xylem vessels (20% reduction), in which the diameter also was reduced by 20 to 30% in hypocotyls and epicotyls. The percentage of vessels colonized by F. oxysporum f. sp. lycopersici was positively correlated to the area under the disease progress curve (AUDPC). However, plants induced with P. oxalicum showed less disease, did not lose the cambium, had a lower number of bundles, and had less vascular colonization by F. oxysporum f. sp. lycopersici (35 to 99%). These effects also were observed in 'Precodor', which is susceptible to races 1 and 2 of F. oxysporum f. sp. lycopersici, and partially in 'Ramón', which is resistant to both races. Renewed or prolonged cambial activity that led to the formation of additional secondary xylem could be one of the reasons for disease reduction in P. oxalicum-induced tomato plants.     

Edaphic Soil Levels of Mineral Nutrients, pH, Organic Matter, and Cationic Exchange Capacity in the Geocaulosphere Associated with Potato Common Scab

ABSTRACT In order to determine possible relationships between geocaulosphere soil properties and severity of common scab of potato caused by Streptomyces scabies, soils were collected from representative commercial potato fields in Canada: in Simcoe and Dufferin Counties, Ontario and across Prince Edward Island (PEI) in August 2004. Soils immediately adjacent to tubers were sampled and analyzed for select edaphic factors and for pathogen presence using polymerase chain reaction (PCR) tests with primers that amplify a region of the TxtA gene involved in regulating the biosynthesis of the thaxtomin toxin family. Individual tubers were assessed visually for scab severity. The relationships between soil chemical factors and disease severity were investigated for each region to detect the strongest relationships. Principal component analysis revealed a distinctive clustering of samples with respect to disease severity in PEI but not in Ontario soils. Total and percent saturation of K (%K) were the only factors found associated with high disease severity in soils from both provinces. In PEI soils, pH, Mg, Ca, Cu, and %K, %Mg, %Ca, and %Na were associated with high disease severity, whereas cation exchange capacity (CEC) and Al were correlated with low disease severity soils. In Ontario, high Mn content was strongly correlated with low disease severity soils, whereas %K and organic matter content were correlated with disease severity. Partitioning samples into presence or absence of the TxtA PCR product with corresponding high or low severity showed further significant relationships in the data. There was an excellent correlation between Streptomyces spp. presence as detected by PCR and disease severity in PEI soils; however, the relationship was not as clear in Ontario soils, where many PCR-positive soils had low disease incidence. Principal component and partial least square analysis indicated that disease severity was predicted by soil factors such as organic matter, CEC, pH, Al, %Ca, %Mg, and %K for PEI but not for Ontario soils. The data reveal that the relationship between scab severity and soil chemical components is complex and potentially soil specific.     

Implication of Systemic Induced Resistance in the Suppression of Fusarium Wilt of Tomato by Pseudomonas fluorescens WCS417r and by Nonpathogenic Fusarium oxysporum Fo47

Fluorescent pseudomonads and nonpathogenic Fusarium oxysporum have been shown to suppress fusarium wilts. This suppression has been related to both microbial antagonism and induced resistance.The aim of the present study was to assess the relative importance of systemic induced resistance in the suppression of fusarium wilt of tomato in commercial-like conditions by a reference strain of each type of microorganism (P. fluorescens WCS417r and nonpathogenic F. oxysporum Fo47). The spatial separation of the pathogen and the biocontrol strains excluded any possible microbial antagonism and implicated the involvement of the systemic induced resistance; whereas the absence of any separation between these microorganisms allowed the expression of both mechanisms. Since systemic induced resistance has often been associated with the synthesis of PR-proteins, their accumulation in tomato plants inoculated with WCS417r or with Fo47 was determined.The analysis of the results indicates that the suppression of fusarium wilt by P. fluorescens WCS417r was ascribed to systemic induced resistance without any detection of the PR-proteins tested (PR-1 and chitinases). In contrast, the suppression achieved by nonpathogenic F. oxysporum Fo47 appeared to be mainly ascribed to microbial antagonism but also to a lesser extent to systemic induced resistance. This induced resistance could be related to the accumulation of PR-1 and chitinases.The possible relationship between the ability of Fo47 to suppress fusarium wilt more efficiently than WCS417r and its ability to show both mechanisms is discussed.