In vivo studies evaluating commercial biofungicide suppression of blight caused by Phytophthora ramorum in selected ornamentals

Phytophthora ramorum is a regulated pathogen in North America, which causes ramorum blight on nursery stock resulting in the implementation of costly quarantine and eradication measures. Earlier studies showed some biofungicides could inhibit growth and reduce the disease in vitro. The objective of this study was to evaluate the effect of commercial biofungicides on in vivo disease development and plant growth on four nursery species when inoculated with three isolates representing each of the NA1, NA2 and EU1 lineages of the pathogen. The plant species were Gaultheria shallon, Rubus spectabilis, Rhododendron caucasicum x R. ponticum var. album and Cornus sericea. Prior to pathogen inoculation, plants were pretreated with Aliette® (standard fungicide), Actigard 50WG Plant Activator®, Actinovate® SP, Sonata®, Serenade®, Plant Helper®, SoilGard® 12G and Pro Mix BX Biofungicide?. A suspension containing 7000 sporangia ml^?1 was used to inoculate plants. Disease severity and foliar biomass were recorded 14 days after inoculation. Actinovate was the only product able to reduce disease severity by about 50% and improve plant growth of the susceptible hosts. Its effectiveness differed by isolate. Several other biofungicides and the standard fungicide provided suppression of disease and improved plant growth but their effectiveness was dependent on host species, product and isolate. None of the products prevented disease and generally the level of control obtained was lower than would be acceptable for a commercial nursery. In addition to assessing the disease control products, it was also observed that rhododendron, salal and salmonberry were highly susceptible hosts of P. ramorum with PR05-001 (NA2) being the most aggressive isolate on them.     

Discovery of a fourth evolutionary lineage of Phytophthora ramorum: EU2

Phytophthora ramorum is a recently introduced, aggressive Phytophthora species that has caused extensive mortality of oak and tanoak trees in the western USA and Japanese larch trees in the UK. P. ramorum is also present on Rhododendron, Camellia, and Viburnum in the nursery industry, which is thought to have been the pathway for its spread into new geographic regions including forests and natural ecosystems. Three lineages of P. ramorum have been described, informally designated EU1, NA1, and NA2, and each lineage is believed to originate from an as yet unknown exotic centre of origin. Preliminary SSR and sequence analysis of isolates from a UK P. ramorum survey revealed seven isolates with profiles that did not match the previously known lineages. Detailed SSR and multilocus sequence analysis of these isolates are presented, allowing us to assign these isolates to a new P. ramorum lineage, designated EU2. Although the known geographical origin of these isolates is currently limited to Northern Ireland and western Scotland, the EU2 lineage isolates have been obtained from four different host plants, including Japanese larch. All isolates are of A1 compatibility type, which implies that this finding does not increase the risk of outcrossing with the EU1 lineage isolates already present in the UK. The oldest EU2 strain was isolated in 2007 but no SSR-based intraEU2 lineage genotypic diversity was detected. The combination of these elements points to a recent introduction, despite emergency phytosanitary measures to control introduction and spread. A PCR-RFLP method for the rapid identification of EU2 lineage isolates is presented.     

Inoculum Density Relationships for Infection of Some Eastern US Forest Species by Phytophthora ramorum

Our objectives were to establish inoculum density relationships between P. ramorum and selected hosts using detached leaf and whole‐plant inoculations. Young plants and detached leaves of Quercus prinus (Chestnut oak), Q. rubra (Northern red oak), Acer rubrum (red maple), Kalmia latifolia (mountain laurel) and Rhododendron ‘Cunningham's White’ were dip‐inoculated with varying numbers of P. ramorum sporangia, and the total number of diseased and healthy leaves recorded following incubation at 20°C and 100% relative humidity. Calibration threshold estimates for obtaining 50% infected leaves based on linear analysis ranged from 36 to 750 sporangia/ml for the five hosts. Half‐life (LD50) estimates (the number of spores for which the per cent of diseased leaves reaches 50% of its total) from asymptotic regression analysis ranged from 94 to 319 sporangia/ml. Statistically significant differences (P = 0.0076) were observed among hosts in per cent infection in response to increased inoculum density. Inoculum threshold estimates based on studies with detached leaves were comparable to those obtained using whole plants. The results provide estimates of inoculum levels necessary to cause disease on these five P. ramorum hosts and will be useful in disease prediction and for development of pest risk assessments.     

Enhanced Recovery of Phytophthora ramorum from Soil Following 30 Days of Storage at 4°C

Chlamydospores of Phytophthora ramorum were used to infest field soil at densities ranging from 0.2 to 42 chlamydospores/cm³ soil. Recovery was determined by baiting with rhododendron leaf discs and dilution plating at time 0 and after 30 days of storage at 4°C, as recommended by USDA‐APHIS. Baiting was slightly more sensitive than dilution plating in recovering P. ramorum immediately following infestation of soil and allowed detection from samples infested with as little as 0.2 chlamydospores/cm³ compared with 1 chlamydospore/cm³ for dilution plating. After 30 days of infested soil storage at 4°C, P. ramorum was detected at significantly (P = 0.05) higher levels than at time 0 with both recovery methods. The results indicate that storage of P. ramorum‐infested soil at 4°C may allow for pathogen activity, such as sporangia production, which may enhance recovery from soil.     

Bacillus spp. and Pseudomonas putida as inhibitors of the Colletotrichum acutatum group and potential to control Glomerella leaf spot

The control of Glomerella leaf spot (GLS) in Brazil is solely based on fungicide sprays and new alternatives are needed. In apple, few biological control methods have been evaluated, and most have focused on post-harvest pathogens. Therefore, the objectives of this work were to study the mode of action of three bacterial strains and the commercial product Serenade® (Bacillus subtilis) against the Colletotrichum acutatum group, the causal agents of GLS, and to evaluate the influence of bacterial isolates and Serenade® on the development of the first cycle of infection disease under controlled conditions. To assess the mode of action of the bacterial isolates against strains of the C. acutatum group, in vitro tests were performed. It was tested the effect of the bacteria on conidial germination and mycelial growth, using three methodologies, (i) fungal-bacterial co-cultivation, (ii) bacterial thermostable metabolites and (iii) bacterial volatile compounds. The influence of the bacterial isolates on the GLS development was assessed using apple seedlings. The seedlings were first sprayed weekly with bacterial suspension for 5 weeks, and were then inoculated with conidia suspensions (10⁴ conidia mL⁻¹) of C. acutatum group isolates. Seedlings were maintained in chambers (CONVIRON) at 25 °C and a 12-h light regime. Disease severity of GLS was evaluated daily by counting typical lesions caused by C. acutatum group on all leaves during 12 consecutive days. The disease progress curve was fitted to nonlinear models for incidence and severity data. The treatments were compared by contrasting epidemiological parameters. Bacillus sp. isolated from the apple phylloplane inhibited more than 60% of the C. acutatum group conidial germination. The mode of action of Bacillus sp. and Bacillus alcalophilus on the C. acutatum group was through the production of fixed and volatile compounds, which inhibited mycelial growth. The primary mode of action of Serenade® on the C. acutatum group was the production of thermostable metabolites capable of completely inhibiting mycelial growth. In the GLS disease cycle, it was possible to adjust the monomolecular model for incidence and the number of lesions. There were significant differences between the epidemiological parameters of GLS in seedlings treated with apple phylloplane bacteria or with Serenade® as compared to the controls, indicating a potential for the use of biological control to manage GLS in apple orchards.     

The Effect of Exposure to Decreasing Relative Humidity on the Viability of Phytophthora ramorum sporangia

Sporangia of three isolates of Phytophthora ramorum representing three different clonal lineages were subjected to relative humidity (RH) levels between 80 and 100% for exposure periods ranging from 1 to 24 h at 20°C in darkness. Plastic containers (21.5 × 14.5 × 5 cm) were used as humidity chambers with 130 ml of glycerine solution added to each container. Glycerine concentrations corresponded to 100, 95, 90, 85 and 80% RH based on refractive index measurements. Sporangia suspensions were pipeted onto nitrile mesh squares (1.5 × 1.5 cm, 15 micron pore size) which were placed in the humidity chambers and incubated at 20°C in darkness. Following exposure periods of 1, 2, 4, 8, 12 and 24 h, mesh squares were inverted onto Petri dishes of selective medium and sporangia germination assessed after 24 and 48 h. At 100% RH, we observed a mean value of 88% germination after 1 h exposure declining to 18% germination following 24 h incubation. At 95% RH, a steeper decline in germination was noted, with means ranging from 79% at 1 h to less than 1% at 24 h exposure. At 90% RH, no germination was noted after 8 or more h exposure, and values were 57%, 22% and 3% germination for the 1, 2 and 4 h exposures, respectively. Germination was only observed at 1 h exposure for both the 85% RH treatment (52% germination) and the 80% RH treatment (38% germination). The three isolates responded similarly over the range of RH values tested. The germination response of P. ramorum sporangia to RH values between 80% and 100% was comparable to that reported for other Phytophthora species. Knowledge of conditions that affect P. ramorum sporangia germination can shed light on pathogenesis and epidemic potential and lead to improved control recommendations.     

Effects of Inoculum Density and Wounding on Stem Infection of Three Eastern US Forest Species by Phytophthora ramorum

Seedlings of three Eastern US forest species Quercus rubra (northern red oak), Quercus prinus (chestnut oak) and Acer rubrum (red maple) were inoculated by applying Phytophthora ramorum sporangia to stems at different inoculum densities with and without wounding. Disease occurred in all treatments involving wounds, and no disease was observed in unwounded treatments. Younger seedlings (2–3 years old) did not differ significantly from older seedlings (5–6 years old) in disease incidence, but older seedlings sustained smaller lesions compared with younger seedlings. For both old and young seedlings, disease on wounded stems was observed down to the lowest sporangia concentration utilized (500 sporangia/ml for old seedlings and 100 sporangia/ml for young seedlings). The results show that in the presence of wounding, even very low sporangia concentrations can result in disease, and further suggest that wounding caused by insects and other factors may play an important role in P. ramorum epidemiology in forest environments.     

Field management of Sclerotinia stem rot of soybean using biological control agents

Biological control agents (BCAs) were evaluated for their efficacy on reducing the number of sclerotia of Sclerotinia sclerotiorum (Lib.) de Bary in the soil and on Sclerotinia stem rot in soybean production systems in Michigan. BCAs included Coniothyrium minitans CON/M/91–08 (Product name: Contans®WG), Streptomyces lydicus WYEC 108 (Actinovate®AG), Trichoderma harzianum T-22 (PlantShield®HC), and Bacillus subtilis QST 713 (Serenade®MAX). At two field locations, soil artificially infested with S. sclerotiorum sclerotia, was treated by incorporating the above BCAs in the topsoil before planting and boscalid was applied as a foliar fungicide at growth stage R1 as a positive control. C. minitans was the most effective BCA and reduced the disease severity index (DSI) by 68.5% and the number of sclerotia of S. sclerotiorum in the soil by 95.3%. S. lydicus and T. harzianum reduced DSI by 43.1% and 38.5% and sclerotia in soil by 90.6% and 70.8%, respectively. B. subtilis only had a marginal effect on S. sclerotiorum. Populations of Bacillus, Streptomyces, Trichoderma spp., and C. minitans collected from soil samples and at 3, 28, 71, and 169 days after BCA application indicated that the population of Streptomyces, Trichoderma spp., and C. minitans did not change significantly throughout the season, which may be the reason for their effectiveness.     

Biological control of clover rot on red clover by Coniothyrium minitans under natural and controlled climatic conditions

The ability of Coniothyrium minitans, contained in the commercial product Contans®WG, to control the development of clover rot in red clover, Trifolium pratense, was for the first time investigated in the field. Studies were performed on an established ley with a grass-clover mixture and on a newly sown pure red clover ley, both at a field site naturally infested with Sclerotinia trifoliorum. In the latter experiment the biocontrol agent was applied either prior to sowing or to growing seedlings. In addition, the ability of sclerotia of two S. trifoliorum isolates to cause disease in detached leaves was studied in a controlled environment. The effect of Contans®WG treatments at temperatures between +5 and +15°C and incubation periods of up to 7 weeks were included. Application of the biocontrol agent to the established ley during early summer, significantly reduced the number of groups of apothecia that developed during autumn in the following year in treated plots, compared to untreated plots. Twice as many red clover plants of the cultivar SW Torun survived in the pure red clover stand experiment the year after Contans®WG application as in the untreated plots, irrespective of how the agent was applied. In the laboratory studies, administering biocontrol treatments to sclerotia significantly reduced disease scores in the detached leaves at all temperatures at an exposure time of 7 weeks. Shorter incubation periods did not always negatively affect sclerotial viability.     

Application of endophytic bacteria for controlling anthracnose disease (Colletotrichum lindemuthianum) on bean plants

Over 100 endophytic bacterial isolates were isolated from surface-sterilised roots of the Fabaceae family in East Azerbaijan farms. These isolates were screened for their in vitro biocontrol activity against Colletotrichum lindemuthianum by dual culture technique using potato dextrose agar (PDA) medium. Eight bacterial isolates (Bacillus subtilis subsp. subtilis, Bacillus atrophaeus, B. tequilensis, B. subtilis subsp. spizizenii, Streptomyces cyaneofuscatus, S. flavofuscus, S. parvus, S. acrimycini) showed promising inhibition on mycelial growth of C. lindemuthianum , and thus, these isolates were selected for greenhouse experiments. The disease control rate using these selected endophytic bacteria was varied from 40 to 76.80% in greenhouse without any negative effects on different growth performance, suggesting that these selected endophytic bacteria are potential to be developed as biocontrol agents.     

Stromata, sporangiomata and chlamydosori of Phytophthora ramorum on inoculated Mediterranean woody plants

Three types of multihyphal structures, stromata, sporangiomata and chlamydosori, are described for the plant pathogen Phytophthora ramorum. Their morphology, morphogenesis and position on the host organ were observed by dissecting, compound and scanning electron microscopy. Stromata were consistently formed one to two weeks after zoospore inoculation of detached leaves and fruits of an assortment of Mediterranean sclerophyll shrubs. Stroma initials appeared subcuticularly or subepidermally and developed as small hyphal aggregates by repeated branching, budding, swelling and interweaving, eventually forming a prosenchyma. They always emerged through the adaxial side of the leaf by rupture of the overlying host tissue. Occasionally sporangia and chlamydosori (packed clusters of chlamydospores) were formed on the stromata. Sporangiomata bore short sporangiophores and clusters of 20–100 sporangia and resembled sporodochia of the mitosporic fungi. The biological significance of these multihyphal structures is discussed. Some epidemiological aspects were also studied: several understorey species of the holm oak (Quercus ilex) woodland were susceptible to in vitro infection with three isolates of P. ramorum originally collected from different ornamental hosts. The risk of spread to this ecosystem is evaluated.     

Distribution of a model biocontrol agent (Serenade® MAX) in apple and pear by mason bees and bumble bees

1. Biocontrol agents (BCAs) are commonly sprayed on flowering pipfruit trees to prevent them from getting infected by various pathogens. By entomovectoring, BCAs can be directly delivered onto the flowers. However, we currently lack knowledge on the distribution dynamics of BCAs by pollinators.
2. Here, managed bees, both bumble bees (Bombus terrestris) and mason bees (Osmia bicornis and Osmia cornuta), were placed in the vicinity of flowering pipfruit trees (pear -‘Conference’, and apple—‘Svatava’ and ‘Jonagold’), and this allowed us to investigate the distribution of a model BCA, namely, Serenade® MAX, from spray-inoculated flowers of a centralized tree to non-inoculated flowers of surrounding receiver trees by bees in an experimental setup in outdoor conditions.
3. One hour after inoculation, we detected an enrichment of BCA in the flowers of the receiver trees and this for each tested pipfruit.
4. The distribution of BCA from treated to untreated flowers was homogenous between the receiver trees for ‘Svatava’, while significantly different loads were detected for both ‘Conference’ and ‘Jonagold’, which might be due to differences in environmental factors, and/or bee characteristics.
5. More research is needed to understand the distribution dynamics of BCAs by pollinators in field conditions, such as in commercial orchards or crop fields, and how this could result in an efficient control.

     

Potential of a new strain of Bacillus subtilis CPA-8 to control the major postharvest diseases of fruit

Biocontrol potential of Bacillus subtilis strain CPA-8 was tested against the main postharvest diseases of orange, apple and stone fruit. Previously, CPA-8 growth was characterized and its antifungal activity in vitro determined against Botrytis cinerea, Monilinia laxa, M. fructicola, Penicillium digitatum, P. expansum, and P. italicum. In vivo activity against these pathogens was tested by treating fruits with cells, endospores or cell free supernatants. CPA-8 treatments cannot control decay caused by P. digitatum and P. italicum on oranges. The higher concentrations of CPA-8 studied were effective in controlling B. cinerea on apple, showing grey mold incidence from 70 to 12.5% in comparison with 100% in the control. However, in general, CPA-8 treatments were not effective in controlling P. expansum. The best results of CPA-8 treatments were obtained in stone fruit against M. laxa and M. fructicola where most treatments resulted in brown rot incidence of 0% compared with 70 and 90% in the control. Based on these results, cultures, cells and cell free supernatants at different concentrations were tested against M. laxa and M. fructicola on stone fruit. Most bacterial concentrations were effective in controlling M. laxa and M. fructicola as well as or better than Serenade® Max, in some treatments showing brown rot incidences of 0% in comparison with 100% of control. Bacterial populations of CPA-8 were maintained stable or increased up to 2-log inside wounds, showing the ability of the bacteria to colonize injured tissues. Experimental evidence suggests that B. subtilis CPA-8 has biocontrol potential for control of postharvest disease on several fruit types.     

Relationship of in vitro and in planta screening: improving the selection process for biological control agents against Fusarium root rot in row crops

Fusarium root rot in row crops is typically managed by cultural practices and fungicide seed treatments. Biological control using microbial agents is another option but needs further development for improved disease management. Screening to identify biocontrol agents are crucial. However, relationships among the steps and how to improve the screening process are unresolved questions. Strains of Burkholderia (4), Bacillus (5) and Trichoderma (26) were studied in vitro against six Fusarium pathogens. All the bacteria and five selected Trichoderma strains were tested in planta in the greenhouse against diseases of Fusarium graminearum and Fusarium oxysporum. Burkholderia ambifaria C628, Bacillus simplex R180, and all Trichoderma isolates showed high reduction in disease levels in corn, soybean and wheat, ranging from 16 to 63%. Responses of the biocontrol agents during in vitro and in planta screening did not always correlate. In vitro and in planta tests should be considered independently in selecting biocontrol candidates.     

Enhanced activity of introduced biocontrol agents in solarized soils and its implications on the integrated control of tomato damping-off caused by Pythium spp

Soil solarization in combination with introduction of biocontrol agents (BCA) was evaluated as a potential disease management strategy for tomato damping-off caused by Pythium spp. A rifampicin resistant Pseudomonas fluorescens strain (PfT-8) and a carbendazim resistant Trichoderma harzianum strain (ThM-1) were introduced into soil following solarization. Tomato seeds were planted into treated field plots. The influence of soil solarization and application of biocontrol agents on damping-off incidence, plant biomass, rhizosphere population of introduced antagonists, and native Pythium spp. was assessed by two consecutive field trials. Damping-off incidence was significantly reduced in solarized plots compared to control. Soil inoculation of biocontrol agents into solarized plots resulted in the highest suppression of damping-off incidence (PfT-8 up to 92%; ThM-1 up to 83%), and increase in plant biomass (PfT-8 up to 66%; ThM-1 up to 48%) when compared to un-solarized control plots. Rhizosphere population of introduced biocontrol agents gradually increased (PfT-8 up to 102% and ThM-1 up to 84%) in solarized soils when compared to unsolarized control. The population of Pythium spp in rhizosphere soil was reduced up to 55% in solarized plots; whereas, application of BCA to solarized soils reduced the rhizosphere population of Pythium spp. by 86 and 82% in P. fluorescens and T. harzianum applied plots respectively.     

Evaluation of fungicides on Indian isolates of Phytophthora colocasiae causing leaf blight of taro

Leaf blight caused by Phytophthora colocasiae is the most destructive disease affecting taro (Colocasia esculenta) worldwide including India. Fungicides (primarily metalaxyl) remain as an important strategy to manage taro leaf blight in India over decades. It is important to monitor isolate sensitivity to identify build-up of fungicide resistance and thereby modify fungicide usage strategies. P. colocasiae isolates representing four different geographical regions of India were evaluated for their sensitivity to metalaxyl and three other commercially available fungicides viz. Samarth, Biofight and Akoton by poisoned media technique. All the isolates tested were sensitive to metalaxyl, nevertheless there is an increase in the effective concentration compared to the previous reports. Among the other fungicides, Samarth was found to be superior in completely inhibiting mycelial growth at 0.05% followed by Biofight at 1%. Metalaxyl and Akoton^® shared a common inhibitory concentration at 2%. The most effective fungicide determined by the in vitro method was evaluated in vivo for studying the pattern of inhibition before and after the disease development in detached taro leaf. The results of the study revealed that build-up on metalaxyl resistance in P. colocasiae is in its course and caution should be taken while administering against taro leaf blight. Fungicide Samarth could be used as an alternative to metalaxyl for management of taro leaf blight.     

Utility of biocontrol agents in the suppression of seed borne pathogenic mycoflora and their effect on seed quality in sorghum (Sorghum bicolor (L.) Moench)

Abstract

The scope of biocontrol agents in the control of sorghum grain molds was investigated both in vitro as well in vivo. Their utility in enhancing the germination and seedling vigor of mold-infected seeds were assessed. The biocontrol agents, Trichoderma viride, T. harzianum, T. hamatum, T. koeningii and Pseudomonas fluorescens (isolates collected from the state of Tamil Nadu, India) were efficient in checking the growth of mold pathogens when grown in Potato Dextrose Agar (PDA) as dual cultures. The bacterial bioagent P. fluorescens was most efficient in checking the growth of all the test pathogens viz., Fusarium moniliforme, Curvularia lunata and Alternaria alternata (66.8, 68.5 and 64.9% inhibition of colony growth respectively) followed by other bioagents for their antagonistic effect. The in vivo effectiveness of the bioagents were enhanced when combined with other components like host-resistance, cultural and chemical control strategies, paving the way for a possible Integrated Disease Management. The percent incidence and severity of molds were lowest (19.44 and 13.5% respectively) with the treatment T₆ (seeds harvested at physiological maturity + propiconazole @ 0.02% + P. fluorescens @ 1 × 10⁸ cfu ml^?1) that recorded maximum 100 seed weight (2.905 g), followed by 2.871 g in T₂ (seeds harvested at physiological maturity + P. fluorescens). The effect of bioagents in enhancing the germination and seedling vigor was most promising. Of all the treatments, P. fluorescens resulted in maximum germination (88%) and seedling vigor (2635.60 vigor index), followed by treatment with T. viride (86.6% and 2478.47 respectively).     

The use of yeasts for biological control of the plant pathogen Sclerotinia sclerotiorum

Yeasts are promising biological control agents(BCAs) for a number of plant diseases. Studieswere carried out to evaluate various adjuvantsand nutrients for their ability to supportgrowth of a yeast BCA (Cryptococcusalbidus). Hydroxyethylcellulose (HEC) andinvert emulsions were found to stimulate growthof C. albidus in vitro. Severalcommercial spray adjuvants were compatible withC. albidus although they did not markedlystimulate growth. Other adjuvants were lethalto the yeast. In controlled environmentand field trials, the yeasts C. albidusand Pichia anomala provided low levels ofcontrol of white mould, a disease of bean (Phaseolus vulgaris) caused by the fungus Sclerotinia sclerotiorum. However, they weregenerally inferior in performance when comparedto either the biocontrol fungus Epicoccumnigrum or to the fungicide iprodione.     

Using combinations of biocontrol agents to control Botrytis cinerea on strawberry leaves under fluctuating temperatures

Experiments were conducted with Botrytis cinerea on strawberry leaves to investigate where combinations of commercially available biological control agents (BCAs) might control B. cinerea more effectively than individual BCAs. Specifically, we studied the persistence of biocontrol activities, spread of BCAs among leaves, and biocontrol efficacy in relation to application regimes: mixed versus single BCA, pre-versus post-inoculation application, and sequential versus simultaneous application. Three BCA products (Sentinel, Serenade and Trianum) were used for this study. Overall, Serenade did not significantly reduce sporulation of B. cinerea on strawberry leaf discs whereas Sentinel and Trianum gave a similar and significant biocontrol efficacy. Biocontrol efficacy remained almost unchanged 10 days after application at 20/20°C (day/night) or 24/16°C temperature regimes. In contrast, reduced biocontrol efficacy at 26/14°C suggests BCA survival was reduced under these conditions. Incidence of B. cinerea sporulation on leaf discs was ca. 60% higher on leaves that emerged after the BCA application than on leaves directly exposed to BCA, indicating insufficient amount of the BCA had managed to spread to new leaves. Combinations of BCAs, whether applied simultaneously or sequentially (48 h apart), did not improve disease control over the most effective BCA within the combination applied alone. This indicated possible antagonism or interference between the BCAs. Results suggested that there was significant antagonism for most combinations of the three BCAs tested and the degree of antagonism increased as the time from BCA application to pathogen introduction lengthened.     

Application of Soil‐borne Actinomycetes for Biological Control against Fusarium Wilt of Chickpea (Cicer arietinum) caused by Fusarium solani fsp pisi

Black root rot, caused by Fusarium solani f.sp. pisi, is a devastating soil‐borne disease in chickpea in Iran with no effective control measures. With the aim of finding applicable biocontrol agents to alleviate the malady, isolates of Actinomycetes isolated from soil and their antagonistic effect against F. solani f.sp. pisi were evaluated both in vitro and in vivo. More than 100 Actinomycetes isolates were screened for their antifungal activities against the pathogen. The most active isolates were evaluated in greenhouse for their biocontrol performance. Based on the results of dual cultures in screening evaluations, the size of inhibition zone of fungal growth, and the most effective antagonist isolates (S3, S12 and S40) were selected for further studies. Identity of active isolates was determined, in this regard, 16S rDNA of isolates were amplified using universal bacterial primers FD1 and RP2. The PCR products were purified and sequenced. Sequence analysis of 16S rDNA was then performed using NCBI BLAST method. Comparison of the near full length 16S rRNA sequence of isolates to GenBank sequences demonstrated that isolates S3 and S12 were most similar to Streptomyces antibioticus, while isolate S40 was most similar to Streptomyces peruviensis. Biocontrol studies of these isolates in control of the disease in greenhouse significantly decreased the disease severity. Actinomycetes isolate S12 demonstrated the greatest effect in reducing disease than the other two. Results of this research are at preliminary stage for developing biocontrol agents. These data can be utilized as a platform for future studies with the aim of commercializing these biocontrol products and hoping to step towards sustainable agriculture.