Identification of rice blast disease-suppressing bacterial strains from the rhizosphere of rice grown in Pakistan

Sixteen bacterial strains isolated from the roots and rhizosphere of rice plants growing in saline and non-saline soils from the Shorkot area of Pakistan were tested for their ability to promote plant growth and reduce the incidence of rice blast disease. When applied to the soil, many of the isolated rhizobacterial strains increased seedling growth and/or suppressed rice blast disease in greenhouse-grown plants of the cultivars Super Basmati and Azucena, but each cultivar responded to different subsets of the bacteria. In the cv Super Basmati, increased blast resistance was correlated with the production of siderophores by the rhizobacteria. Several strains inhibited the growth of the causative agent of rice blast disease, the fungal pathogen Magnaporthe grisea, in an in vitro dual culture assay. Direct bioantagonism was correlated with disease resistance in Super Basmati, but not in Azucena, and direct antagonism as a cause for the reduced disease incidence is also unlikely since no epiphytic colonisation of leaves was detected. Rhizosphere colonisation by the bacteria in plants grown in sterile sand was correlated with disease resistance in Super Basmati, but not in Azucena. As well as the differences in strains that protected each cv against blast disease, we also found that there were differences in the ability of some strains to protect plants against blast depending on soil type. Hence, there are complex interactions between rhizobacteria and rice plants with respect to biocontrol of rice blast disease, dependent upon both rice cv and soil type. The identity of strains that promoted high levels of disease protection, including three that performed well across all plant cultivars and growth conditions, was determined by 16S rRNA gene sequencing.     

Occurrence of rice blast (Magnaporthe oryzae) and identification of potential resistance sources in Uganda

Rice blast caused by Magnaporthe oryzae continues to be the most destructive disease of rice worldwide, and is a number one disease of rice in Uganda. We present the footprints of M. oryzae importance, distribution, incidence and severity in the rice growing agro-ecologies of Uganda for 2009/2010, and the potential mitigation measures. Our data show that rice blast affects more than 50% of the cultivated rice area on average, and ranks as the most important disease encountered in the field. Between and within agroecologies, both incidence and severity varied (P < 0.05) reflecting the contribution of different cropping practices on rice blast epiphytotics. The highest blast incidence and severities were recorded in Bugiri, Butaleja, Mbale and Lira farmlands, which are the ancestral rice cultivation areas in Uganda. These areas showed mean yield reductions of >30% relative to other locations, suggesting that rice production history played a significant role in rice blast outbreaks. Broadcasting and drill seeding yielded 42.4% less than transplanted rice. Growing two rice crops per year is one of the methods suggested to increase rice production in Uganda, but there was a higher disease incidence in the late season than in the early season, indicating the need for improved resistant varieties. Four blast resistance genes (Pi9, Piz-t, Pi19 and Piz-5) and the cultivar Tetep had the lowest (≤4) blast severity scores in all the test locations. It could be suggested that these genes are potential resistance sources for developing varieties, which would be more relevant for the double cropping systems.     

Assessing the effect of resistant-susceptible associations and determining thresholds for associations in suppressing leaf and neck blast of rice

Blast has become a major problem in the upland rice of Indonesia, as the improved resistant varieties with high yield performances released in the 90's became susceptible only a few years after their release. The present study investigated the efficiency of association of susceptible and resistant varieties in reducing leaf and neck blast and increasing yield. Two sets of experiments were conducted in fields with associations through using different proportions of resistant variety, and two types of susceptible variety (moderately and very susceptible) in the association. The efficiency of the association was measured by comparing disease intensity and yield in the variety when grown in association and when grown as a pure stand. ANOVAs and logistic regressions indicated that resistant-susceptible variety association was more efficient in reducing neck blast than leaf blast. Associations were more efficient (1) when the proportion of resistant variety was increased, (2) when a moderately susceptible variety was used instead of a very susceptible variety, and (3) when disease level in the pure stand was higher. Associations did not penalize the performances of the resistant variety, neither in terms of disease intensity, nor yield. Logistic regressions allowed to estimate the probability of a given association to reduce disease (or to increase yield) above a preset threshold, according to the proportion of resistant variety and to the type of susceptible variety.     

空间诱变育成抗稻瘟病和白叶枯病水稻突变体浙101

 早籼浙9248经卫星搭载诱变处理，种子的发芽率及当代植株的性状均没有明显的变化，但对秧苗的生长表现出一定的刺激作用；SP2在株高、生育期、穗长、每穗粒数、千粒重、结实率及抗病性等性状上出现了较大的分离。经病区多代筛选培育成的突变体浙101，在熟期、抗病性和产量等性状上比原亲本有明显改良，2001~2002年经浙江省多点稻瘟病和白叶枯病联合鉴定，叶瘟平均级分别为1.4和1.7级，最高级为5.0级；穗瘟平均级分别为1.6和1.3级，最高级为3.0级；抗谱频率分别为70%和60%；对白叶枯病抗性平均级为1.4级，最高级为5.0级。抗病性比原亲本和对照有显著提高。试验表明，浙101是一个高抗稻瘟病兼抗白叶枯病的水稻突变体，可作为水稻品种改良的新抗源。     

A set of near-isogenic lines for blast resistance genes with an Indica-type rainfed lowland elite rice (Oryza sativa L.) genetic background

A set of near-isogenic lines for blast resistance genes was developed by using an Indica-type elite rice variety, IR49830-7-1-2-2, suitable for the rainfed lowland conditions in the tropics, as a genetic background. Initially, we revealed that IR49830-7-1-2-2 harbors five blast resistance genes - Pia, Pib, Pik-s, Pita, and Pi11(t) - by using a differential system involving 19 selected standard blast isolates from the Philippines. Based on this result, we developed nine near-isogenic lines (NILs) targeting eight resistance genes - Pik, Pi7(t), Pi3, Pi5, Pita-2, Piz-5, Pish, and Pi9 - by recurrent backcrossing. The introgression of each resistance gene in the NILs was confirmed by reaction patterns to the blast isolates, allelism tests, and DNA marker analysis. In addition, a genome-wide DNA marker survey revealed that most of the chromosome regions in each NIL were of the IR49830-7-1-2-2 type. The agricultural characteristics of most of the developed NILs were almost the same as those of IR49830-7-1-2-2. Moreover, with one exception, they showed submergence tolerance similar to IR49830-7-1-2-2. The developed NILs could be used as a multiline variety suitable for the rainfed lowland in the tropics.     

Canopy temperature on clear and cloudy days can be used to estimate varietal differences in stomatal conductance in rice

Leaf photosynthetic potential, a major determinant of yield potential in rice, is mostly explained by stomatal conductance (g_s) as well as leaf N content (Rubisco protein content). Therefore, the rapid and precise evaluation of g_s as well as leaf N content would be a valuable addition to rice breeding programs aimed at improved yield potential. We established a simple method based on infrared thermography to estimate varietal differences in g_s. Seven rice varieties were cultivated in two-row plots in 2007, and canopy temperatures in three varieties, including a control variety, Koshihikari, were simultaneously measured to determine canopy temperature difference (CTd) between Koshihikari and the other varieties on clear days. The varieties Takanari, Habataki, and TUAT1-5-6a displayed significantly (P < 0.05) lower leaf temperature and higher g_s and leaf photosynthetic rate (P_n) than Koshihikari. CTd was closely correlated with g_s and P_n. These results indicate that CTd may be useful as a relative index to estimate varietal differences in g_s. A significant CTd was still observed even under cloudy conditions in 2008. Takanari and TUAT1-5-6a had lower leaf temperature than Koshihikari on cloudy days as well as on clear days. Subsequent investigation of photosynthetic light response curves revealed that Takanari and TUAT1-5-6a had higher g_s and P_n than Koshihikari under lower irradiance. These results suggest that infrared thermography may be a simple method of evaluating varietal differences in g_s through CTd, and that it may be practicable even under cloudy conditions.     

Wilt disease management and enhancement of growth and yield of Cajanus cajan (L) var. Manak by bacterial combinations amended with chemical fertilizer

Root nodulating Sinorhizobium fredii KCC5 and Pseudomonas fluorescens LPK2 were isolated from nodules of Cajanus cajan and disease suppressive soil of tomato rhizosphere, respectively. Both strains produced IAA, siderophore, solubilized insoluble phosphate, showed chitinase and β-1,3-glucanase activities, and strongly inhibited the growth of Fusarium udum. It also caused degradation and digestion of cell wall components, resulting in hyphal perforations, empty cell (halo) formation, shrinking and lysis of fungal mycelia along with significant degeneration of conidia. LPK2 produced volatile cyanogen (HCN). Combinations of S. fredii KCC5 and P. fluorescens LPK2 with half dose of chemical fertilizers showed a significant increase in seed germination (94%) while seed germination with co-inoculated strains (KCC5 + LPK2), KCC5 and LPK2 alone was 90, 84 and 82% respectively as compared to control 77%. After 120 days of sowing, per plant number of pods, nodules, shoot length, root length, shoot weight and root weight were greater for the combination with half dose of chemical fertilizers compared to the control. Combinations of S. fredii KCC5 and P. fluorescens LPK2 with half dose of chemical fertilizers resulted in an 82% increase in grain yield per hectare compared to the control. Both strains KCC5 and LPK2 led to proto-cooperation as evidenced by synergism, aggressive colonization of the roots, and enhanced growth, suggesting potential biocontrol efficacy against Fusarium wilt in C. cajan.     

稻秆还田对水稻主要病害发生的影响

通过水稻秸秆还田小区试验,初步探讨了多年秸秆还田对15个水稻品种病害的影响。试验结果表明：秸秆还田的老稻田15个水稻品种的稻瘟病、鞘腐病、纹枯病的病情指数都高于对照稻田的病情指数。黑龙江省农垦总局建三江分局主栽品种空育131,叶瘟病情指数增加了2．3％,穗颈瘟增加了2．6％,鞘腐病增加了2．2％,纹枯病增加了0．4％。     

Management of the root-knot nematode Meloidogyne incognita on tomato with combinations of different biocontrol organisms

The nematicidal effect of Pseudomonas fluorescens, Paecilomyces lilacinus, Pichia guilliermondii and Calothrix parietina singly or in combination was tested against root-knot nematode, Meloidogyne incognita. Treatments with P. fluorescens and P. lilacinus caused mortality of M. incognita as 45% and 30% of juveniles after 48 h of exposures, respectively compared to water control in vitro. Under greenhouse conditions, all treatments reduced the disease severity and enhanced plant growth compared to untreated control. Application of P. fluorescens, P. lilacinus and P. guilliermondii Moh 10 was more effective compared to C. parietina. There was a negative interaction between C. parietina and either P. lilacinus or P. guilliermondii. Fresh and dry weight of shoots and roots of plants were significantly reduced as a result of infection with M. incognita, however application of biocontrol agents singly or in mix recovered this reduction. Moreover, they enhanced the growth parameters compared with the control. Our results proved that application of different biocontrol agents (P. fluorescens, P. lilacinus and P. guilliermondii) not only has a lethal effect on nematode, but also enhances the plant growth, supplying many nutritional elements and induction the systemic resistance in plants. Presence of C. parietina as a soil inhabitant cyanobacterium could antagonize biocontrol agents leading to the reduction of their practical efficiency in soil.     

A novel method for controlling rice blast disease using fan-forced wind on paddy fields

Rice blast disease is one of the obstacles of rice production not only in Japan but throughout rice producing countries. The effects of fan-forced wind on the incidence of rice blast disease were studied in two successive seasons. Electric fans (5 KW, 110-cm blade diameter) set on the ridge of paddy fields at a height 5 m from the ground level were used to artificially generate wind. In season 1, the fan operated twice daily for 30 min periods at 11:00 pm and 4:00 am from June 15 to September 1. The blocks of the paddy fields were divided into 6 zones according to wind speed and distance from the fan. The wind speed ranged from 2.0 to >7.3 m/s. The incidence of both rice leaf and panicle blast was significantly lower in the zones receiving wind between 2.6 and 7.3 m/s; however, the zone that received a velocity >7.3 m/s was severely affected by leaf and panicle blast. The zone that received the a wind speed of 2.6 m/s or lower exhibited an inefficient reduction of leaf and panicle blast disease, but it was better than in the control fields. In season 2, a wind-forced fan was applied from June 16 to September 9, with the velocity adjusted between ca. 3.0 and 6.0 m/s. The incidence and severity of leaf and panicle blasts (chuff, rachis-branch and neck blast) were efficiently reduced in the wind-treated fields. Fan-forced wind was more effective than the application of conventional chemical fungicides for controlling rice blast disease. These data demonstrate the potential of fan-forced wind for controlling rice blast disease.     

A novel,integrated method for management of witches' broom disease in Cacao in Bahia,Brazil

A three-year field study was conducted in Bahia, Brazil to validate several strategies for management of witches' broom disease in cacao caused by Moniliophthora perniciosa. Treatments which were applied alone or in combination included applications of biological control fungus Trichoderma stromaticum, fungicide copper hydroxide and phytosanitary broom removal. When compared with untreated control treatments, higher pod yields and consistently lower pod losses were obtained by alternating fungicide with biocontrol application. Pod losses caused by witches' broom were also reduced by fungicide treatment or by phytosanitary broom removal when applied alone or in combination, however total pod production per tree was consistently low whenever broom removal was used as a management strategy. While application of biocontrol fungus alone was not able to reduce witches' broom on pods, it reduced vegetative broom formation and also increased the number of pod-forming flower cushions. The present study indicates that alternating fungicide copper hydroxide with biocontrol fungus T. stromaticum without expensive phytosanitary broom removal is not only a better disease management strategy in Bahia but also results in better yields and thereby better net economic returns.     

Management of Cephaleuros parasiticaus Karst (Trentepohliales: Trentepohliaceae), an algal pathogen of tea plant,Camellia sinsensis (L) (O. Kuntze)

The efficacy of certain biocontrol agents were evaluated against Cephaleuros parasiticus Karst, a causal organism of red rust disease in tea plants. Spraying of systemic fungicides was found to be superior to biocontrol agents followed by algicides in controlling the disease under field condition. Dipping of shears in the solutions of fungicide, detergent or biocontrol agents failed to protect the disease spread. The study on the impact of different harvesting practices on disease development showed the highest disease incidence in continuously shear harvested fields and least in hand plucked fields. There was a reduction in disease incidence in spraying of a solution containing urea and murate of potash mixture at 1% level. The maximum green leaf yield and productivity index were recorded in biocontrol agents treated plots. Among the various group of biocontrol agents evaluated, Streptomyces sannanensis and Streptomyces griseus belong to actinomycetes was better than bacterial and fungal antagonists in terms of disease protection and increase in yield potential. Similarly, tea bush canopy architecture, physiological and biochemical parameters were also increased in plants treated with biocontrol agents. In the case of untreated control plots, the disease incidence was increased from 36% to 44.5% and the plants were found unhealthy in terms of chlorosis, stunted growth and heavy flowering with banji buds. The shoots collected from treated and untreated control plots were subjected to manufacture CTC black tea which revealed almost all the tea quality parameters were significantly improved in the treated plot over the untreated plots.     

Insecticidal effect of recombinant endophytic bacterium containing Pinellia ternata agglutinin against white backed planthopper, Sogatella furcifera

White backed planthopper (WBPH; Sogota furcifera Horvath) has become the major threat to rice crops throughout Asia, damaging plants both through its feeding behavior and by acting as a virus vector. Here, we developed a novel method for biologically controlling WBPH by using endophytic bacterium to express anti-pest plant lectins. Strain SJ-10 of an endophytic bacterium, characterized as Enterobacter cloacae by morphological, physiological, biochemical and 16s rDNA characteristics, was isolated from rice seedlings. The Pinellia ternate agglutinin (PTA) gene was cloned into SJ-10 for expression. The positive transformant, selected by antibiotic resistance, was evaluated using PCR, SDS-PAGE and Western blot assay. After inoculation, rSJ-10 could colonize rice plants so that they expressed PTA, and then the rice was shown to have insecticidal activity against WBPH. The results showed that rSJ-10 could significantly decrease the survival and fecundity of WBPH fed on rice seedlings (p < 0.01). At day 19, the fecundity of WBPH inoculated with rSJ-10, or with wild-type SJ-10 was decreased by 86.1%, and 25.6%, respectively. At day 22, numbers of WBPH on rice in the control were 19.4 times greater than on rice inoculated with rSJ-10. At day 26, the rice seedlings all died in the control group, but the seedlings inoculated with rSJ-10 grew well. The results showed that the rice seedlings inoculated with rSJ-10 expressing PTA protein were endowed with the anti-pest activity against WBPH. Further work is needed to investigate whether the rice plants expressing rPTA are toxic to mammals. This research highlights a way to biologically control planthoppers by recombinant endophytic bacteria expressing plant lectins.     

Effect of plant growth promoting rhizobia on seed germination, growth promotion and suppression of Fusarium wilt of fenugreek (Trigonella foenum-graecum L.)

Five bacterial strains (TR1 to TR5) isolated from root nodules of fenugreek (Trigonella foenum-graecum) were tested for their plant growth promotory traits and biocontrol potential against Fusarium oxysporum. On the basis of morphological, physiological, biochemical and molecular characteristics, strains TR1 and TR3 - TR5 were identified as Ensifer meliloti, and TR2 as Rhizobium leguminosarum. All bacterial isolates utilized phosphate in vitro. Except TR5, all isolates produced IAA and none of them showed volatile cyanogens production. Except TR3, all isolates produced in vitro siderophore. Isolate TR1 and TR4 showed chitinase production while only TR2 showed β-1,3-glucanase activity. Isolates TR1, TR2 and TR5 exhibited ACC deaminase activity. Isolates TR1, TR2 and TR4 inhibited the growth of F. oxysporum, causing loss of structural integrity of the mycelium, hyphal perforation, lysis, fragmentation and degradation. The potential for nodulation and nitrogen fixation of the strains were confirmed by amplification of 500 bp nodC and 781 bp nifH fragments. The application of the TR1 + TR2 combination resulted in increased grain yield by 35% and 36% of fenugreek in two consecutive field trials, respectively as compared to control. Maximum increments in vigour index, nodule number and root and shoot biomass were recorded with seed inoculated with consortium (TR1 + TR2) followed by single inoculation as compared to control. The antibiotic resistant marker strain of E. meliloti TR1^strep+ and R. leguminosarum TR2^tet+ confirmed the efficient colonization of fenugreek roots. This study showed that these rhizobial isolates have properties of biocontrol agents and may be applied to promote the growth of fenugreek.     

不同施氮方式对黑龙江省水稻主栽品种稻瘟病发生的影响

通过不同施氮方式对黑龙江省主栽品种稻瘟病的研究,探讨了施用氮肥对稻瘟病发病规律的影响。试验结果表明:稻瘟病的发生氮肥起着决定性作用,N肥高、中、低及0氮肥条件下,叶瘟和穗颈瘟的发病规律呈现出相同的趋势,随着N肥量的增多发病率增强,病情严重;不同的施肥方式叶瘟和穗颈瘟也呈现出相同的规律,基蘖肥与穗粒肥比为7:3发病率与病情指数最高,发病最严重,基蘖肥与穗粒肥比为8:2、6:4表现病情相对较低。随着密度的降低,各品种发病情况无规律性。     

Effectiveness of plant extracts on suppression of damping-off and wilt diseases of lupine (Lupinus termis Forsik)

The aim of this study was to evaluate water and organic solvent of plant extracts for protection of lupine plants against damping-off and wilt diseases caused by Fusarium oxysporum f. sp. lupini, F. oxysporum f. sp. lupini Snyder & Hansen was isolated from diseased lupine roots collected from different locations of Minia, Assiut and New Valley governorates. Water leaf extracts of Calotropis procera, Nerium oleander, Eugenia jambolana, Citrullus colocynthis, Ambrosia maritime, Acacia nilotica and Ocimum basilicum and fruit extracts of C. colocynthis, C. procera and E. jambolana reduced damping-off and wilt diseases caused by F. oxysporum f. sp. lupini. Water extracts of E. jambolana leaves, C. colocynthis fruits and N. oleander leaves were the most effective ones for controlling such diseases. In contrast, A. nilotica and O. basilicum extracts were the least effective ones. Organic solvent extracts of tested plants reduced damping-off and wilt diseases than water extracts. Butanolic and ethereal extracts were highly effective in reduction of diseases than the other tested extracts. Under field conditions, during winter growing seasons 2008/09 and 2009/10, ethereal and butanolic extracts of N. oleander and E. jambolana leaves and C. colocynthis fruits significantly reduced the percentage of wilt severity as well as improved growth parameters (plant height, number of branches, pods and seeds per plant) and increased seed index, total seed yield/hectare compared with control treatment, while protein content in seeds was not effected. In conclusion, our study demonstrated that some plant extracts can be used for biocontrol of lupine damping-off and wilt diseases.     

Biological control of Macrophomina phaseolina by chemotactic fluorescent Pseudomonas aeruginosa PN1 and its plant growth promotory activity in chir-pine

Ten strains of Pseudomonas aeruginosa (PN1 ˜ PN10) isolated from rhizosphere of chir-pine were tested for their plant growth promontory properties and antagonistic activities against Macrophomina phaseolina in vitro and in vivo. P. aeruginosa PN1 produced siderophore, IAA, cyanogen and solubilized phosphorus, besides producing chitinase and β-1,3-glucanase. In dual culture, P. aeruginosa PN1 caused 69% colony growth inhibition. However, cell free culture filtrate also posed inhibitory effect but to a lesser extent. After 90 days, P. aeruginosa PN1 increased plant growth and biomass in pots trial containing M. phaseolina-infested soil. PN1 showed the strong chemotaxis toward root exudates resulting in effective root colonization. Moreover, increased population in rhizosphere of these bacteria was also recorded after 90 days of treatment. Thus, chemotactic fluorescent P. aeruginosa PN1 exhibited strong antagonistic property against M. phaseolina, suppressed the disease and improved plant growth of the seedlings of chir-pine proving potential biocontrol agent.     

水稻应用不同药剂对稻瘟病防治效果分析

通过对2%加收米、75%稻艳、80%多菌灵、三环唑、25%咪鲜胺5种药剂进行稻瘟病的田间防效对比试验,各个处理 分别喷施4遍.结果表明,75%稻艳可湿性粉剂防治效果最好,叶瘟防治效果达到47.7%,穗颈瘟的防治效果达到51.1%.     

Rice Blast Resistance of Transgenic Rice Plants with Pi-d2 Gene

Resistance to rice blast of transgenic rice lines harboring rice blast resistance gene Pi-d2 transformed from three different expression vectors of pCB6.3kb, pCB5.3kb and pZH01-2.72kb were analyzed. Nine advanced-generation transgenic rice lines with Pi-d2 gene displayed various resistance to 39 rice blast strains, and the highest disease-resistant frequency reached 91.7%. Four early-generation homozygous transgenic lines with Pi-d2 gene exhibited resistance to more than 81.5% of 58 rice blast strains, showing the characteristic of wide-spectrum resistance. The transgenic embryonic calli selected by the crude toxin of rice blast fungus showed that the callus induction rate of immature embryo from transgenic rice plants decreased as the concentration of crude toxin in the culture medium increased. When the concentration of crude toxin reached 40%, the callus induction rate of immature embryo from transgenic lines was 49.3%, and that of the receptor control was 5%. The disease incidence of neck blast of the transgenic rice lines in fields under induction was 0% to 50%, indicating that the rice blast resistance of transgenic rice lines is much higher than that of the receptor control.     

Antifungal activity of polyacetylenes isolated from Cirsium japonicum roots against various phytopathogenic fungi

Antifungal substances from a methanol extract of Cirsium japonicum roots were purified and characterized, and their antifungal activities against various plant pathogens were evaluated. Three polyacetylene substances were isolated from roots of C. japonicum using repeated column chromatography; these were identified as ciryneol A, ciryneol C and 1-heptadecene-11,13-diyne-8,9,10-triol by mass and nuclear magnetic resonance spectral analyses. In vitro antifungal activity of the three substances varied according to compound and target species. Magnaporthe oryzae, Colletotrichum coccodes, Colletotrichum acutatum, Pythium ultimum and Botrytis cinerea were relatively sensitive to the three polyacetylenes, with IC₅₀ values below 50 μg mL⁻¹. In vivo, they all showed similar and broad antifungal spectra against the seven plant diseases tested. At 500 μg mL⁻¹, all three compounds effectively suppressed the development of rice blast, rice sheath blight, tomato late blight, wheat leaf rust and red pepper anthracnose, with control values over 90%. They were highly active especially against wheat leaf rust; they controlled the development of this disease more than 88% even at a concentration of 125 μg mL⁻¹. In addition, ciryneol C effectively suppressed barley powdery mildew. This is the first report on the antifungal activities of the three polyacetylenes from roots of C. japonicum against plant pathogenic fungi. Polyacetylenes from roots of C. japonicum may contribute to the development of environmentally safer alternatives to protect crops from various phytopathogenic fungi.