Prevention,with Trichoderma harzianum Rifai aggr., of reinfestation by Sclerotium rolfsii Sacc. and Rhizoctonia solani Kühn of soil fumigated with methyl bromide,and improvement of disease control in tomatoes and peanuts

Application of Trichoderma harzianum Rifai aggr. after soil fumigation with methyl bromide improved the control of Sclerotium rolfsii Sacc. and Rhizoctonia solani Kühn in a peanut field. Although soil fumigation controlled the diseases caused by these pathogens, it was followed by rapid reinfestation by S. rolfsii and R. solani. The biological control agent T. harzianum prevented reinfestation of the fumigated soil by the pathogens (88% reduction) both in a controlled environment and in field conditions. In soil treated with T. harzianum, survival of sclerotia was considerably less than in the untreated control. The combined treatment, of fumigation and T. harzianum applications, caused almost total mortality of sclerotia in soil in the laboratory and in the field. Application of T. harzianum to the root zone of tomatoes effectively controlled S. rolfsii in a field naturally infested with S. rolfsii and R. solani. Transplanting plants treated with T. harzianum into soil fumigated with methyl bromide reduced disease incidence by 93% and increased yield by 160%.     

Superior bio-efficacy of a combined formulation of carbendazim and mancozeb in inducing defense responses in chilli seedlings against Sclerotium rolfsii Sacc. in comparison with methyl jasmonate

The objective of this work was to investigate the efficacy of a prepackaged combined formulation, Companion (carbendazim 12% + mancozeb 63% WP), sole application of carbendazim 50% WP, manozeb 75% WP and methyl jasmonate (MeJA), an inducer of systemic acquired resistance on disease severity and their role in post-infectional defense responses in chilli seedlings against Sclerotium rolfsii. Seeds were treated for 8 h with MeJA (2.5 mM and 5.0 mM) and each of fungicides (500 ppm), and were sown in pots containing soil and fungal inocula (95:5 w/w). At 15 days after sowing maximum defense against fungal infection was exhibited by Companion comparably followed by the sole application of carbendazim and mancozeb. MeJA reduced percent mortality of S. rolfsii-infected chilli seedlings significantly as compared to the inoculated control. Assessment of peroxidase (POX) and esterase (EST) at 15 days after sowing revealed the increased activity under inoculated condition. Highest POX activity in MeJA treatments (5 mM > 2.5 mM) was followed by the Companion treatment. Highest EST activity was registered in Companion treatment. The zymogram of POX isozymes showed over-expression of POX 2 and POX 4 isoforms, and induction of POX 1 isoform in inoculated treatments. On the other hand, that of EST isozymes showed induction of EST 1 isoform in Companion, carbendazim and MeJA treatments. All EST isoforms were over-expressed in Companion-treated seedlings. Both fungicides and MeJA showed significant effects on disease severity, induction of defense enzymes and isozyme pattern in S. rolfsii-infected chilli seedlings. Contact and systemic fungicides under the experiment demonstrated differential responses. The combination formulation was superior in disease control to application of the fungicide components individually. They compared favourably with MeJA in induction of defense-related enzyme activities. All these findings are new with respect to the chilli-S. rolfsii host–pathogen interaction system, S. rolfsii representing the sclerotial basidiomycetes in particular.     

Growth promotion and induction of resistance in tomato plant against Xanthomonas euvesicatoria and Alternaria solani by Trichoderma spp.

In tomato crop, the induction of resistance emerges as an important alternative for achieving the reduction of chemicals in disease control. This study aimed to evaluate the ability of 28 Trichoderma isolates to promote the growth of tomato seedlings and to induce systemic resistance (ISR) against Xanthomonas euvesicatoria and Alternaria solani, the causal agents of bacterial spot and early blight, respectively. Twelve isolates promoted the increase of plant dry matter mass (DMM) above 100%, showing the great potential of these strains. All isolates were able to colonize the root system of tomato plants. The plant growth-promoting isolates were further evaluated for potential elicitation of ISR. Treatment of the soil with all Trichoderma isolates provided protection in tomato plants from 24.13 to 95.94% against X. euvesicatoria and 30.69 to 95.23% against A. solani. The most efficient isolates in reducing the severity of bacterial spot and early blight were the isolates IB 28/07, IB 30/07, IB 37/01 and IB 28/07, IB 30/07 and IB 42/03, respectively. The effect of different time intervals between Trichoderma application and inoculation with pathogens in inducing systemic resistance in tomato plants was evaluated for the isolate IB 28/07. IB 28/07 conferred protection against both diseases at all time intervals, confirming the ability of the isolate to reduce the severity of these diseases up to 21 days after treatment of tomato plants. In vitro assays revealed that all isolates of Trichoderma were able to degrade cellulose. Only the isolate IB 34/08 showed antagonistic activity against X. euvesicatoria and none caused reduction in the in vitro mycelial growth of A. solani. Trichoderma isolates were identified at species level by DNA sequencing.     

Combined use of Streptomyces sp. A6 and chemical fungicides against fusarium wilt of Cajanus cajan may reduce the dosage of fungicides required in the field

Biological control agents offer one of the best alternatives to reduce the use of pesticides. This investigation studied the tolerance to fungicides and integrated use of the potential biocontrol agent Streptomyces sp. A6 for control of Fusarium wilt of pigeon pea, Cajanus cajan. Streptomyces sp. A6 exhibited strong tolerance towards most of the fungicides used in the study at concentrations higher than those recommended for field applications. The isolate showed enhanced growth and mycolytic enzyme production in the presence of sulphur, mancozeb, carbendazim, fosetyl aluminium and triadimefon. The fungicides mancozeb, sulphur and carbendazim were selected for further studies. Effective concentrations (EC₅₀ values) of the test fungicides that reduced Fusarium spore germination and fungal biomass by 50% were determined. Similarly, the EC₅₀ for inhibiting fungal spore germination and reducing fungal biomass to 50% by Streptomyces sp. A6 and culture filtrate (CF) were also determined. Combining the EC₅₀ dose of the culture and CF with test fungicides was found to be more effective for controlling Fusarium infection in C. cajan compared to the sum of the effects of the individual treatments. Such combined use of biocontrol agent with fungicides can reduce the dosage of toxic fungicides in agricultural fields, thereby reducing environmental risks. Tolerance and synergistic interaction of Streptomyces sp. A6 with frequently used fungicides suggested its potential in integrated pest management. To the best our knowledge, this is the first extensive study on integrated use of Streptomyces species with fungicides.     

Actinomycetes mediated biochemical responses in tomato (Solanum lycopersicum) enhances bioprotection against Rhizoctonia solani

Six actinomycetes isolates, namely Streptomyces toxytricini vh6, Streptomyces flavotricini vh8, S. toxytricini vh22, Streptomyces avidinii vh32, Streptomyces tricolor vh85 and vh41, an isolate of an unknown species of Actinomycetales, were tested for their efficacy in protecting tomato (Solanum lycopersicum) against Rhizoctonia solani under green house conditions. Actinomycetes treated plants showed better growth in terms of high chlorophyll content, higher phenylalanine ammonia lyase (PAL) activity and high total phenolic content. Qualitative and quantitative estimation of phenolic compounds from tomato leaves showed significant accumulation of six phenolic acids, gallic (29.02 μg g⁻¹ fresh leaf wt), ferulic (11.44 μg g⁻¹ fresh wt), cinnamic (56.84 μg g⁻¹ fresh wt), gentisic (24.19 μg g⁻¹ fresh wt), chlorogenic acid (1.72 μg g⁻¹ fresh wt) and salicylic (0.39 μg g⁻¹ fresh wt) acid, in actinomycetes treated plants. Biochemical profiling, when correlated with plant mortality in actinomycetes treated and untreated plants, indicated that isolates vh6 and vh8 offered 44.55% and 40.14% disease reductions, respectively compared to the control. These results established that these organisms have the potential to act as biocontrol agents.     

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.     

Maize genotype susceptibility to Rhizoctonia solani and its effect on sugar beet crop rotations

Root and crown rot is the major soil-borne fungal disease in sugar beet. In Europe, the disease is mainly caused by the anastomosis group (AG) 2-2IIIB of the basidiomycete Rhizoctonia solani (Kühn). No chemical fungicide to control the disease has been registered in Europe. Therefore, agronomic measures must be optimized to keep the disease severity below an economic damage threshold and to minimize white sugar yield losses. R. solani AG 2-2IIIB infects many other crops besides sugar beet, including maize, where it causes root rot. Sugar beet and maize are frequently grown in the same crop rotation. The proportion of cultivated maize in several European sugar beet growing areas is expected to rise due to a projected increase in demand for renewable resources over the next few years. Although the susceptibility to and tolerance of the disease varies among cultivars in both crops, little is known about the effects of cultivar susceptibility in the pre-crop on a subsequent susceptible crop. The cultivation of R. solani-resistant maize genotypes in rotation with resistant sugar beet might therefore be a useful tool in an integrated control strategy against R. solani, eliminating the need to restrict the desired crop rotation for phytosanitary reasons. A crop rotation experiment with artificially inoculated R. solani was conducted in the field to investigate the pre-crop effects of maize cultivars which differed in their susceptibility to R. solani on a susceptible sugar beet cultivar. We hypothesized that the maize genotype would influence the inoculum potential and performance of a susceptible sugar beet genotype grown after a maize pre-crop, and that this would correlate with the susceptibility of the maize genotype. The results demonstrate that the susceptibility of maize genotypes is consistent over a period of years and that cultivated maize genotypes influenced the inoculum potential measured as disease severity in sugar beet. However, disease severity in sugar beet did not correlate with the disease susceptibility of the genotype of the maize pre-crop. Possible reasons for this missing relationship might be differences in the quality of maize residues for the saprophytic survival of the pathogen or a genotype-specific alteration of the antagonistic microbial community. However, our findings showed that in the presence of maize- and sugar beet-pathogenic R. solani, the most favourable maize cultivar for a crop rotation cannot be determined solely on the basis of its resistance level against Rhizoctonia root rot.     

Suppressiveness of steam-exploded biomass of Miscanthus sinensis var. giganteus against soil-borne plant pathogens

Commercial compost is a renewable resource widely used in horticulture as an organic amendment, though its suppression against soil-borne plant pathogens remains limited. Preliminary studies conducted on the disease suppression effect of the biomass waste obtained in a steam explosion plant demonstrated positive results. Steam-Exploded Biomass (SEB) of Miscanthus sinensis var. giganteus, a herbaceous perennial energy crop, is a multifunctional renewable energy resource, which could also be useful in crop protection to find valid alternative to the compost use in horticulture. The purpose of this work was to assess the suppressiveness of SEB against five plant pathogenic fungi that are important in many Italian horticultural cropping systems. Analyses of the microbial inhibitors (furfurals, organic acids and lignosulfonates) present in the SEB were performed by the High Performance Liquid Chromatography technique. Assessment of toxic effect of the furfurals present in the SEB (furfuraldehyde and 5-hydroxymethylfurfural), added to a growing medium at the different concentration ranges, was carried out in vitro on Phytophthora nicotianae, Pythium ultimum, Fusarium oxysporum f. sp. lactucae, F. oxysporum f. sp. melonis and Rhizoctonia solani. The suppressiveness of SEB, added to a potting soil used in horticulture at the different doses, was tested in vivo on tomato/P. nicotianae, cucumber/P. ultimum, lettuce/F. oxysporum f. sp. lactucae, melon/F. oxysporum f. sp. melonis and bean/R. solani. The results showed that furfuraldehyde, 5-hydroxymethylfurfural, lignosulfonates, acetic and formic acid were detected at a concentration of 2.93, 0.28, 4.12, 10.07 and 1.88 g/kg SEB, respectively. The P. nicotianae, P. ultimum and R. solani fungi were highly inhibited by the addition of 3.2 g/L furfuraldehyde and 0.48 g/L 5-hydroxymethylfurfural. Moreover, the inhibitory effect was found not adequate against F. oxysporum at the same concentrations. The SEB increased significantly the suppressiveness level of the peat substrate on P. ultimum in cucumber and R. solani in bean in all the trials. For P. nicotianae in tomato, the SEB addition showed a significant suppression at the 20 and 30% doses, but the change was not significant at the 10% dose. In case of F. oxysporum f. sp. lactucae in lettuce and F. oxysporum f. sp. melonis in melon, the SEB addition showed no suppressive effect with respect to compost in all the trials. In conclusion, the SEB could be used against some soil-borne fungal diseases in place of compost in the potting soil, and its suppressiveness could be related to the concentration of the microbial inhibitors produced during the processing of fresh biomass in the steam explosion plant.     

Population dynamics of Xanthomonas campestris pv. vitians on different plant species and management of bacterial leaf spot of lettuce under greenhouse conditions

The population dynamics of Xanthomonas campestris pv. vitians (Xcv) was studied both externally and internally in lettuce, tomato and pepper plants. In addition, the application of bactericides during transplant production period was carried out for the management of bacterial leaf spot of lettuce under greenhouse conditions. Epiphytic populations of Xcv were recovered on leaves of lettuce plants (10⁵ CFU/g) 5 weeks after sprayed than the other plant species when inoculated with 10⁸ CFU/ml of Xcv. When plants of each crop species infiltrated with the bacterium at 10⁵ CFU/ml, the highest populations were developed in lettuce (10⁸ CFU/cm²) followed by pepper with 10⁶ CFU/cm² and tomato with 10⁵ CFU/cm² 10-days after infiltration. Application of a mixture of Maneb and Kocide or Kocide alone as a foliar spray on lettuce significantly reduced the incidence and disease severity of bacterial leaf spot by 29 and 27% respectively. Spread of the bacterium and development of the disease may partly be managed by avoiding intercropping of these plants commonly grown in close proximity to lettuce. For the management of leaf-associated populations of Xcv in lettuce, use of a mixture of Maneb and Kocide is advocated to minimize the effect of attacks.     

Activities of essential oils from Asarum heterotropoides var. mandshuricum against five phytopathogens

Some secondary metabolites of plants function as antimicrobial products against phytopathogens and constitute an increasingly important class of pesticides. In the present study, the essential oil of Asarum heterotropoides var. mandshuricum was analyzed by GC/MS and its antimicrobial activity was evaluated against five phytopathogenic fungi. Major components of the oil were methyleugenol (59.42%), eucarvone (24.10%), 5-allyl-1,2,3-trimethoxybenzene (5.72%), and 3,7,7-trimethylbicyclo(4.1.0)hept-3-ene (4.93%). The essential oil and the most abundant component, methyleugenol, were separately assayed for inhibition of 5 pathogens: Alternaria humicola, Colletotrichum gloeosporioides, Rhizoctonia solani, Phytophthora cactorum and Fusarium solani. Both the oil and methyleugenol strongly inhibited the growth of the test pathogens (IC₅₀ values <0.42 μg ml⁻¹) except F. solani, with the best activity against P. cactorum (IC₅₀ values = 0.073 and 0.052 μg ml⁻¹, respectively). It is concluded that the essential oil of A. heterotropoides var. mandshuricum has a broad antiphytopathogenic spectrum, and that methyleugenol is largely responsible for the bioactivity of the oil. The mode of action of methyleugenol against P. cactorum is discussed based on changes in the mycelial ultrastructure.     

Mycoparasitic Trichoderma viride as a biocontrol agent against Fusarium oxysporum f. sp. adzuki and Pythium arrhenomanes and as a growth promoter of soybean

Trichoderma viride was proved as an effective biocontrol agent against two fungal pathogens, Fusarium oxysporum f. sp. adzuki and Pythium arrhenomanes, infecting soybean. During an in vitro biocontrol test, Trichoderma showed mycoparasitism and destructive control against the tested fungal pathogens. Both the pathogens significantly influence the germination and P. arrhenomanes had a severe effect (only 5% germination). The root system of the soybean plant was poorly developed due to the infection and it exerted a negative influence on the nodulation and further growth phases of the plant. During pot assay along with biocontrol activity, Trichoderma showed growth promoting action on the soybean plant. Trichoderma enhanced growth of shoot and root systems and fruit yield after 12 weeks of growth. Pythium and Fusarium infected plants treated with Trichoderma had ∼194% and 141% more height than pathogens alone. The fruit yield treated with Trichoderma was ∼66 per plant whereas the yield was only 41 for a control plant. The plants infected with Pythium and Fusarium and treated with Trichoderma had fruit yields of 43 and 53 respectively and those were 5 and 1.6 times higher than plants infected with pathogens.     

Control of tomato powdery mildew (Leveillula taurica) in the Comarca Lagunera, Coahuila State, Mexico, supported by the spray forecast model Tomato.PM

Tomato powdery mildew [Leveillula taurica (Lev.) Arm.] is a common disease of tomato crops in the Comarca Lagunera region of the state of Coahuila, Mexico. To control the disease, fungicides are commonly sprayed up to 18 times on a weekly basis in this region. Effectiveness of the spray forecast model Tomato.PM, developed for California conditions, to reduce fungicide spraying, was evaluated. Disease dynamics on plants treated with three different fungicides (azoxystrobin, myclobutanil and wettable sulfur), with and without model recommendations, was analyzed in three experiments at three locations. When spraying followed model recommendations, it was possible to eliminate 11-16 fungicide applications with no significant effect on fruit yield. All three fungicides reduced the severity of disease but their effects varied with the cropping cycle. A significant increase in total yield due to fungicide spraying was observed in one out of three experiments.     

Control of Rhizoctonia solani fruit rot of tomatoes by Trichoderma harzianum Rifai

Application of Trichoderma harzianum, to soil or by coating tomato fruits, reduced Rhizoctonia solani fruit rot by up to 43% and 85%, respectively, under laboratory conditions. When mixed with naturally infested soil, Trichoderma reduced R. solani inoculum potential by 86% in field trials. It also significantly reduced fruit rot by 27–51%.     

Study on antagonistic effects of Talaromyces flavus on Verticillium albo-atrum, the causal agent of potato wilt disease

Talaromyces flavus a fungal antagonist, was isolated from soil samples collected from potato fields in Varamin and Karaj districts, Tehran province, Iran. Antagonistic effects of T. flavus isolates against Verticillium albo-atrum, the causal agent of potato wilt disease were investigated in the laboratory and greenhouse conditions. T. flavus colonies were recovered after three weeks from soil samples cultured on selective medium. Antagonistic effects of volatile and non-volatile extracts of T. flavus isolates on V. albo-atrum growth were investigated in the laboratory and five that caused higher growth inhibition of V. albo-atrum, were selected for greenhouse experiments. Infection index was compared in the greenhouse in a split plot trial with five isolates applied to soil, seed, or both arranged in a randomized complete block design with four replications. The minimum infection index was observed when seed were treated with T. flavus with the most effective isolate being Tf-Po-V-52. On seed, the minimum infection index was observed with Tf-Po-V-50. The most effective T. flavus isolate was also evaluated in a field experiment. Results indicated that infection index in seed treatment contained this isolate was 0.15 whereas that of control was 3.5. The overall results of this study showed that it may be possible to manage potato Verticillium wilt disease effectively by using T. flavus, a biocontrol fungus.     

Repellent effects of five monoterpenoid odours against Tribolium castaneum (Herbst) and Rhyzopertha dominica (F.) in Calabar, Nigeria

Secondary plant compounds are recognised as important components of plant defence system against herbivores and pathogens. Five monoterpenoids, (R)-linalool, 1,8-cineole, (S)-2-heptyl acetate, (S)-2-heptanol and citral, which are natural components of the essential oils of Aframomum melegueta (K. Schum) and Zingiber officinale (Roscoe), were tested at the ratios in which they occur naturally for repellent activity against Tribolium castaneum (Herbst.) and Rhyzopertha dominica (F.) in a 4-way olfactometer. The results showed the repellent properties of the compounds as both beetles spent less time in the olfactometer arm containing the test stimuli. (R)-linalool and (S)-2-heptanol were stronger repellent compounds than the others. Linalool showed good repellent activity against T. castaneum (P = 0.001) as the insect spent 1.22 min in the test arm compared to the control arms (2.78 min), and R. dominica (P = 0.001) with 0.89 min in the test arm compared to 2.87 min in the control arms. With (S)-2-heptanol, T. castaneum spent 1.23 min in the test arm compared to 2.83 min in the control arms. R. dominica spent 1.61 min in the test arm and 2.69 min in the control arms. For the number of entries or visits made, while both insects were significantly repelled (P < 0.05) by the linalool-treated arm than the control, only R. dominica was repelled by the (S)-2-heptanol-treated arm (P = 0.038) compared to the control arms. The results indicate that A. melegueta and Z. officinale essential oils and their components could be suitable as safer repellents or fumigants against T. castaneum and R. dominica.     

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.     

Field evaluation of Sclerotium rolfsii, a biological control agent for broadleaf weeds in dry, direct-seeded rice

The fungal pathogen Sclerotium rolfsii isolate SC64 is being assessed as the basis for a mycoherbicide for biological control of broadleaf weeds in dry-seeded rice fields. Species tested for susceptibility in the field included Cyperus difformis, Lindernia procumbens, Rotala rotundifolia, Ammannia baccifera and Eclipta prostrata. Following preliminary small plot field applications in summer 2008 and 2010, applications of fungus-infested solid substrates (mixture of rice hulls and bran) of 60-140 g m⁻² were conducted at two sites, Nantong and Rugao, in Jiangsu province, China in summer 2010. The sites included a one-year fallow field and a wheat-rice rotation field. Plant mortality was recorded 7 and 14 days after inoculation (DAI). Percentage mortality ranged from 50 to 89% and 30-71% in the 2008 and 2010 solarium small plot trials, respectively. At the Nantong site field trial, 30-60% plant mortality and 31-59% fresh weight reduction were recorded at 14 DAI when applied for the first time but the efficacy increased to 39-86% and 42-90% for plant mortality and fresh weight reduction at 14 DAI with a repeated application. Higher levels of plant mortality (42-77%) and fresh weight reduction (52-82%) were achieved at 14 DAI at the Rugao site with a single treatment, due to the lower weed density and more favourable temperature and humidity conditions at the time of pathogen application. Results confirmed that S. rolfsii SC64 is a potential biocontrol agent of some of the broadleaf weeds tested in dry-seeded rice.     

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.     

Evaluation of fungicides to control Cylindrocarpon liriodendri and Cylindrocarpon macrodidymum in vitro, and their effect during the rooting phase in the grapevine propagation process

The efficacy of 14 selected fungicides against Cylindrocarpon liriodendri and Cylindrocarpon macrodidymum was evaluated in vitro by testing their effect on mycelial growth and conidial germination. Carbendazim, hydroxyquinoline sulphate, imazalil, and prochloraz were the most effective fungicides in reducing mycelial growth in both Cylindrocarpon species. Captan, copper oxicloride, didecyldimethylammonium chloride and thiram were the most effective to inhibit conidial germination of both species. A pot assay was also conducted with captan, carbendazim, copper oxychloride, didecyldimethylammonium chloride, hydroxyquinoline sulphate, imazalil and prochloraz in order to determine their potential to prevent infections caused by C. liriodendri and C. macrodidymum during the rooting phase in the grapevine propagation process. All fungicides significantly decreased the root disease severity values in both species compared with control treatment, with the exception of imazalil in C. macrodidymum. In the case of the percentage of reisolation, all values were lower than those obtained for the control treatment, but only captan, carbendazim and didecyldimethylammonium chloride were significantly different in the case of the cuttings inoculated with C. liriodendri, and prochloraz in the case of those inoculated with C. macrodidymum.     

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.