Biological control of root-rot of Coleus forskohlii Briq. using microbial inoculants

The root-rot or wilt of Coleus forskohlii is a very serious soil-borne disease caused by Fusarium chlamydosporum. A field study was undertaken to study the possibility of controlling the disease using three biocontrol agents viz., Glomus mosseae, Pseudomonas fluorescens, Trichoderma viride, singly and in combination. Planting of coleus cutting was done in wilt sick soil. Inoculation with Trichoderma viride + Glomus mosseae gave the best result in controlling the disease. The same treatment also resulted in maximum growth, yield and root forskolin concentration of coleus. Plants treated with T. viride + G. mosseae showed a disease severity index of 33.28% compared to uninoculated control plants, which had a maximum disease severity index of 85.5%. The fungicide Emisan (0.2%) was not as effective as the biocontrol agents in controlling the pathogen.     

Bioagent based integrated management of Phytophthora blight of pigeonpea

Phytophthora blight (Phytophthora drechsleri f. sp. cajani) is one of the major yield limiting factors of short duration varieties of pigeonpea (Cajanus cajan). For eco-friendly and sustainable management of the disease, four antagonists, Pseudomonas fluorescens, Bacillus subtilis, Trichoderma viride and T. hamatum, were evaluated under in vitro and in vivo conditions. P. fluorescens produced maximum inhibition zone, while Trichoderma species overgrew on the host mycelium and caused lysis. The fungicides apron (metalaxyl), ridomil MZ (metalaxyl + mancozeb), capton (captaf), difolatan (captafol), thiram (tetramethylthiuram disulphide) and bavistin (carbendazim) at both 0.3 and 0.6% were found to be compatible with P. fluorescens while B. subtilis was compatible only with apron at 0.3%. T. viride grew and sporulated well at 0.3 and 0.6% apron and 0.2% ridomil MZ. The bioagents proved to have potential and those which were compatible under in vitro were evaluated under field conditions. The efficacy of fungicide in combination with bioagents was greater compared to fungicide alone. The integration of P. fluorescens with apron or ridomil MZ as seed treatment significantly reduced the Phytophthora blight incidence and enhanced seed germination and grain yield.     

Biological control of onion leaf blight disease by bulb and foliar application of powder formulation of antagonist mixture

Abstract

Three antagonists: Pseudomonas fluorescens (Pf1), Bacillus subtilis and Trichoderma viride, were tested alone and in combination for suppression of onion leaf blight (Alternaria palandui) disease under glasshouse and field conditions. The average mean of disease reduction was 24.81% for single strains and 42.44% for mixtures. In addition to disease suppression, treatment with a mixture of antagonists promoted plant growth in terms of increased plant height and ultimately bulb yield. Though seed treatment of either single strain or strain mixtures alone could reduce the disease, subsequent application to root, leaves or soil further reduced the disease and enhanced the plant growth. The mixture consisting of Pseudomonas fluorescens Pf1 plus Bacillus subtilis plus Trichoderma viride was the most effective in reducing the disease and in promoting plant growth and bulb yield in greenhouse and field tests.     

Potential for integrated biological and chemical control of damping-off disease caused by Pythium ultimum in tomato

Pythium ultimum (Trow) is one of the main causes of damping-off disease in many parts of the world. Control of the disease depends mainly on application of chemical fungicides. However, soil treatments with fungicides are not always feasible due to economical and ecological reasons. Soil-borne, non-pathogenic bacteria of the genus Pseudomonas fluorescens with the ability to antagonise fungal phytopathogens, represent a realistic alternative to chemical fungicides and show great promise with respect to protect plant roots from fungal-induced diseases. In an attempt to find an integrated control system of damping-off disease in tomato, fungicides including azoxystrobin, metalaxyl-M and pyraclostrobin were applied alone and in combination with P. fluorescens isolate CW2. The fungicides were tested in in vitro for their antagonistic potential against P. ultimum and for compatibility with CW2. It was found that the fungicides were fungitoxic to P. ultimum, but did not inhibit the growth of the P. fluorescens. The efficacy of the fungicides alone and in combination with CW2 was also tested in greenhouse experiments against damping-off disease on tomato. Two concentrations (5 and 10?μg?ml^?1) were applied. Damping-off incidence of tomato seedlings in Humosoil^?:sand mixture infested with P. ultimum was reduced following seed treatment with the fungicides. However, the degree of control obtained varied significantly depending on the fungicide used. Combined seed treatment with P. fluorescens and the fungicides resulted in a significant improvement in disease control and improved plant growth as indicated by shoot and root dry weights. Metalaxyl-M treatment applied alone or in combination with P. fluorescens, significantly protected tomato seedlings against damping-off. Strobilurin fungicides stimulated plant growth compared to metalaxyl-M. Combined treatment of tomato seeds with strobilurin fungicides and CW2 showed a moderate to good disease control and an increase in shoot and root dry weights.     

Management of sorghum grain mold through induced resistance mechanism using foliar spray of Pseudomonas fluorescens strains and non-conventional chemicals: a field study

Abstract

The study involved in-vitro and field studies using Pseudomonas fluorescens strains and some non-conventional chemicals for sorghum grain mold management. The culture filtrate of P. fluorescens strains PF-1 and PF-2 inhibited spore germination (p?<?0.001) and mycelial growth of grain mold fungi. Also, tested chemicals and commercial fungicides showed significant mycelial growth inhibition (p?<?0.001). PF-1, PF-2, ZnSO₄·7H₂O, salicylic acid and propiconazole were potentially effective and hence were tested in field (by foliar spraying). Two set of experiments were conducted simultaneously using naturally and artificially (Fusarium challenged) induced infected sorghum panicles. The lowest infection (score 4.2) was recorded with propiconazole. Disease severity was well controlled by PF-1 and PF-2, compared to ZnSO₄·7H₂O and salicylic acid. Furthermore, disease control was validated by determining total phenolic, flavonoid content and differentially induced phenolic acids (HPLC analysis) in harvested grains. Additionally, results of biochemical-traits, compatibility studies and antibiotic sensitivity of strains were found to be favourable.     

Control of powdery mildew in okra using cultural filtrates of certain bio-agents alone and mixed with penconazole

This study was carried out to evaluate the culture filtrates of certain bio-agents (Epicoccum nigrum, Epicoccum minitans, Epicoccum sp., Trichoderma harzianum, Trichoderma viride and Bacillus pumilus) alone and mixed with penconazole against powdery mildew in okra. The results showed that the culture filtrate of E. nigrum induced the highest efficacy against powdery mildew relative to other treatments in both tested seasons. Moreover, culture filtrate of E.nigrum mixed with the tested fungicide gave higher efficiency against powdery mildew than use of the fungicide alone. The efficacy of the tested culture filtrates against powdery mildew due to the presence of different antifungal compounds identified by (GC–MS) analysis. This study suggests the ability of using the culture filtrates of the tested bio-agents as alternative of fungicides to control powdery mildew in okra. Furthermore, the results implied the possibility to minimise the use of fungicides by mixing with the microbial culture filtrates.     

No impact of neonicotinoids on male solitary bees Osmia cornuta under semi‐field conditions

The ubiquitous use of agrochemicals is one driver for the ongoing loss of insect biomass and diversity. Data show that field‐realistic concentrations of neonicotinoid insecticides can negatively affect both population density and the fitness of solitary bees. However, the underlying mechanisms for these effects remain poorly understood. Here, using an established semi‐field experimental set‐up and Osmia cornuta as a solitary bee model, we examined the effects of field‐realistic concentrations of a common neonicotinoid insecticide (clothianidin) on male larvae and adults. Besides measuring lethal (i.e., overwintering success and adult survival) and sub‐lethal endpoints (i.e., emergence mass and emergence duration), we examined, for the first time, potential effects on the male reproductive physiology of a solitary bee (i.e., sperm quantity and viability). The data revealed no significant effects on any of the measured response variables. This may be due to the low degree of clothianidin exposure (0.56 ng g^?1) and/or the apparent low susceptibility of solitary bee larvae to neonicotinoids. Furthermore, it is conceivable that ideal foraging conditions, combined with optimal weather and lack of other environmental stressors, may have improved the ability of bees to cope with the insecticide. To reliably assess and understand the environmental hazards of agrochemicals, a holistic approach, including laboratory, semi‐field and field data is required. Knowledge of underlying mechanisms will help to mitigate the current global declines of insect populations.     

Evaluating the promise of Trichoderma and Anabaena based biofilms as multifunctional agents in Macrophomina phaseolina-infected cotton crop

The influence of biofilmed formulations composed of Trichoderma viride and Anabaena torulosa as matrices was investigated in Macrophomina phaseolina (Tassi) Goid.-infected cotton crop, in terms of plant growth and biocontrol parameters. Trichoderma based biofilms were developed with Azotobacter chroococcum, Pseudomonas fluorescens and Bacillus subtilis, while A. torulosa biofilms were prepared using B. subtilis and T. viride as components. Scanning electron microscopy revealed significant colonisation of biofilms on the root surface, which could be correlated with lowest mortality of 5.67%, recorded using T. viride–B. subtilis biofilm. An increase of 4–7% in polyphenol oxidase enzyme activity was recorded in all biofilm-treated samples, particularly those in which B. subtilis was present. The highest value of 1360.22 µg microbial biomass carbon g^?1 soil was recorded in Anabaena–B. subtilis biofilm treatment. Significantly higher values of plant and soil nutrient parameters in treatments in which biofilms were used vis-à-vis individual cultures reveal their promise. Such novel biofilmed biofertilizers with multiple useful traits can be beneficial options for effective nutrient and pest management of cotton crop.     

Evaluation of Some Fungi and Bacteria for Potential Control of Safflower Rust

Some fungal and bacterial isolates applied as soil and seed treatments in greenhouse trials gave a good protection against Puccinia carthami Cda. on safflower, significantly reducing the infection from rust-infested seeds. Trichoderma viride and T. harzianum added alone and in combination as air-dried inoculum to the soil were the most effective fungal isolates. However T. viride at two higher rates reduced the rate of emergence of safflower seedlings. Among bacteria, Bacillus subtilis, B. cereus, isolates of Pseudomonas fluorescens and B. thuringiensis reduced rust infection when added both as soil drench and as seed treatment. Good results in the biocontrol of P. carthami were also obtained with some combination antagonist treatments such as T. viride + B. cereus, T. viride + P. fluorescens (16), T. viride + T. harzianum + B. cereus and T. viride +, T. harzianum + P. fluorescens (16). Fungal isolates added as soil treatment increased seedling length, whereas no similar effects were observed when these isolates were applied as seed treatment.     

Sequential testing scheme for the assessment of the side-effects of plant protection products on the predatory bug Orius laevigatus

This paper describes a number of test methods, to beused in a sequential scheme, for testing the side-effects ofplant protection products on anthocorid bugs. Orius laevigatuswas used as test species. A `worst case' laboratory method wasdeveloped for evaluating the effect on mortality of the nymphsand the reproduction of the adults. An extended laboratory methodgives information on the effect of pesticides on adults undermore natural conditions. A semi-field test is executed in smallplots under field (glasshouse) conditions. Finally a field testsimulates the conditions in a commercial crop. For harmfulchemicals, both a laboratory and a semi-field persistence testwas developed to determine the safety period after which thepredatory bugs can be re-introduced in the greenhouse withoutbeing adversely affected. The total result of this sequentialscheme should indicate whether, and with what restrictions, aparticular pesticide can be implemented in IPM programs in whichO. laevigatus is used for control of western flower thrips(Frankliniella occidentalis). Twenty-two pesticides were testedincluding five fungicides, twelve insecticides and fiveacaricides. The fungicides captan, carbendazim, sulphur, thiram,tolylfluanid and the insecticides or acaricides pymetrozine,pyriproxyfen, tebufenozide and hexythiazox were harmless in the`worst case' laboratory test and thus did not need furthertesting. Imidacloprid, diafenthiuron, lufenuron, tebufenpyrad,abamectin, pyridaben and bifenthrin were harmful in thesemi-field test and should, in the tested concentrations, betternot be used simultaneously with the predatory bug. Theinsecticide dichlorvos was found harmful but short-lived in thepersistence test; it should be used before the introduction ofthe predatory bug O. laevigatus only when a safety period of 7days is respected. Pirimicarb was slightly toxic in thesemi-field test but short-lived. In IPM programs this aphicideshould be used at a low application frequency.     

Evaluation of some fungicides for seed treatment and foliar application in management of damping-off of seedlings and blight of rapeseed caused by Alternaria brassicae

Eighteen fungicides were first evaluated for their effects on growth of Alternaria brassicae and for ascertaining their fungicidal and fungistatic natures in artificial cultures. The chemicals emerging fungicidal in action, were later evaluated for their efficacy as seed treatment and foliar application in the management of damping-off of seedlings and blight of rapeseed separately. Of 18 fungicides tested, six fungicides, viz., Dithane M-45, Dithane Z-78, Ziram, Difolatan-80, Blitox-50 and Benlate completely inhibited the growth of the pathogen and were fungicidal in action. Thiram and Brestan-60, which also caused total growth inhibition, were, however, fungistatic. Benlate (0.1 %) followed by Dithane M-45 was best seed-dressing fungicide for controlling damping-off of seedlings. Dithane M-45 (0.2%) followed by Dithane Z-78 as foliar spray was most effective for controlling the blight and increasing the yield in field trials.     

Antifungal potentiality of some plant extracts against Fusarium sp.

Abstract

Aqueous extracts of 46 plants belonging to 32 different families of the plant kingdom were screened for antifungal activity against eight important species of Fusarium viz., Fusarium equiseti, F. moniliforme, F. semitectum, F. graminearum, F. oxysporum, F. proliferatum, F. solani and F. lateritium. The test fungi were isolated from maize, paddy and sorghum seeds collected from Mysore district, Mysore, India. Among the several plants screened only 12 plants have recorded significant antifungal activity. The antifungal activity of aqueous extracts varied among the test pathogens and was compared with that of the synthetic fungicides Blitox, Captan, Dithane M-45 and Thiram. F. proliferatum, which showed high susceptibility for the aqueous extracts, was tested using different solvent extracts viz., petroleum ether, benzene, chloroform, methanol and ethanol extracts of all the 12 plants. The results revealed that these plants could be exploited for ecofriendly management of the diseases caused by the test fungal pathogens and seed biodeterioration during storage.     

Three native Pseudomonas fluorescens strains tested under growth chamber and field conditions as biocontrol agents against damping-off in alfalfa

Alfalfa (Medicago sativa) is one of the most important crops used in Uruguay for livestock feeding. Seedling diseases, particularly damping-off, are a critical factor which limits its establishment. Three native Pseudomonas fluorescens strains, UP61.2, UP143.8 and UP148.2, previously isolated from Lotus corniculatus, were evaluated to determine their efficacy as biological control agents for alfalfa seedling diseases in the field. Their compatibility with the alfalfa-Sinorhizobium meliloti symbiosis was also assessed. In growth chamber conditions seed inoculation with Pseudomonas strains did not affect different parameters of alfalfa-rhizobium symbiosis as shown by nodulation rate and shoot dry weight of plants. The presence of the commercial inoculant strains of S. meliloti did not impair colonization by the P. fluorescens and vice versa. In field trials the dynamics of rhizobial rhizospheric populations were not affected by the presence of P. fluorescens. Each P. fluorescens strain successfully colonized alfalfa roots at adequate densities for biocontrol activity. Results showed that P. fluorescens strains provided a 10–13% increase in the number of established plants relative to the control, an intermediate result compared to the fungicide treatment (24%). The alfalfa above-ground biomass was increased by 13% and 15–18% in the presence of the fungicide and P. fluorescens strains, respectively. Therefore, results from this study demonstrated that the three P. fluorescens strains provided effective control against soil-borne pathogens and suggest a potential use in the development of a commercial inoculant to be applied for the control of legume seedling diseases.     

In vitro and in vivo antagonism of biocontrol agents against Colletotrichum lindemuthianum causing bean anthracnose

Bean anthracnose caused by Colletotrichum lindemuthianum is a serious seed borne disease. For devising an effective management strategy, the efficacy of different bioagents, viz. Trichoderma viride, Trichoderma harzianum, Trichoderma hamatum and Gliocladium virens conducted under in vitro and in vivo conditions revealed maximum inhibition of mycelial growth in dual culture (59.48%) and inverted plate (55.98%) with T. viride. All the bioagents overgrew the pathogen and the principal mechanism of mycoparisitism observed was coiling, brusting and disintegration of pathogen hyphae. Culture filtrate from T. viride was found best as it completely inhibited radial growth at 25 and 50% concentration and reduced the spore germination of test fungus significantly. However, lower concentrations of culture filtrate from all bioagents showed little effect on spore germination. Seed application of bioagents was found better as compared to soil application. A maximum increase in seed germination and inhibition of seed borne infection was observed with T. viride followed by T. harzianum under pot culture conditions. T. viride has the maximum potentiality to suppress the spore germination, mycelial growth, seed borne infection of C. lindemuthianum and increased seed germination when compared with the other biocontrol agents.     

Effect of combination between inducer resistance chemicals and systemic fungicides on management of sweet melon downy mildew

The efficiency of some inducer resistance chemicals (IRCs) like bion, chitosan, humic acid and salicylic acid as well as the fungicides like Folu-Gold, Galben Copper, Previcure-N and Redomil Gold Mancozeb on management of sweet melon downy mildew, caused by Pseudoperonospora cubensis was evaluated in vitro and in vivo. Also, the efficiency of the alternation between the sprayed two fungicides and IRCs on management of the disease and the produced fruit yield and its total soluble solids (TSS) under field conditions were assessed. The inhibitory effect of the IRCs and the tested fungicides on sporangial germination of P. cubensis resulted in a significant reduction in the germinated sporangia. In addition, IRCs were less effective than the fungicides. Disease management revealed the same trend of the in vitro experiment when they sprayed fungicides on sweet melon plants artificially inoculated with the sporangia of the causal fungus under greenhouse conditions. Furthermore, under field conditions, spraying sweet melon plants with the two tested fungicides was the most efficient in decreasing the disease and increasing fruit yield and its TSS, to somewhat, followed by the alternation between them and the tested IRCs. In addition, IRCs treatments showed the lowest efficiency in this regard.     

Antagonistic assessment of Trichoderma spp. by producing volatile and non-volatile compounds against different fungal pathogens

Trichoderma spp. are well-known biological agents that have significant antagonistic activity against several plant pathogenic fungi. In the present study, Trichoderma spp. were tested in vitro for their antagonistic activity against different spp. of Fusarium and Alternaria viz. Alternaria alternata, A. brassicae, A. solani, Fusarium oxysporum and F. solani using dual plate assay and by the production of volatile and non-volatile compounds. The results obtained revealed that Trichoderma harzianum and T. viride effectively inhibited the growth and spore production of different spp. of Fusarium and Alternaria. The highest growth inhibition was found in A. alternata 62.50% and 60.00% by non-volatile compounds of T. harzianum and T. viride, respectively. Similarly, the volatile compounds inhibit the maximum growth of A. alternata 40.00% and 35.00% by T. harzianum and T. viride, respectively. Volatile and non-volatile compounds of Trichoderma spp. were analysed by GC-MS technique and the properties of distinguished compounds showed antifungal, antimicrobial and antibiotic activities. Volatile compounds of T. harzianum and T. viride showed highest percent abundance for glacial acetic acid (45.32%) and propyl-benzene (41.75%), respectively. In case of non-volatile compounds, T. harzianum and T. viride showed D-Glucose, 6-O-α-D-galactopyranosyl- (38.45%) and 17-Octadecynoic acid (36.23%), respectively. The results of present study confirmed that T. harzianum can be used as a promising biological control agent against Alternaria and Fusarium spp. that cause diseases in various vegetables and crops.     

Rhizobacterial bioformulation for the effective management of Macrophomina root rot in mungbean

Abstract

Fluorescent pseudomonads based bioformulation was evaluated for their ability to control Macrophomina root rot disease in mungbean (Vigna mungo). P. fluorescens isolate Pf1 showed the maximum inhibition in mycelial growth of Macrophomina phaseolina under in vitro conditions. Bioformulation of Pf1 with chitin was effective in reducing the root rot incidence in green gram both under glasshouse and field conditions. The rhizosphere colonization of P. fluorescens was observed appreciable with the green gram plants. However, Pf1 amended with chitin colonized effectively. Furthermore, the induction of defence-related enzymes and chemicals in plants by Pf1 amended with or without chitin and neem were tested. Increased accumulation of defence enzymes viz., phenylalanine ammonia lyase (PAL), peroxidase (PO), polyphenol oxidase (PPO), chitinase, β-1,3-glucanse and phenolics were observed in Pf1 bioformulation amended with chitin, pre-treated plants challenge inoculated with M. phaseolina under glasshouse conditions. The present study reveals that in addition to direct antagonism and plant-growth promotion, PGPR strains amended with chitin bioformulation induced defence-related enzymes and pathogenesis related (PR) proteins which collectively enhance the resistance in green gram against the infection of M. phaseolina.     

Susceptibility of Various Stages of Trichogrammatoidea armigera Nagaraja to Some Pesticides and Effect of Residues on Survival and Parasitizing Ability

The toxicity, persistence and effect on parasitism of 10 insecticides, eight fungicides and one acaricide on Trichogrammatoidea armigera Nagaraja, an egg parasitoid of a Helicoverpa armigera (Hb), were investigated in the laboratory and under field conditions. At field recommended dosages, the fungicides oxycarboxin, copperoxychloride, streptomycin sulphate + tetracycline hydrochloride and 2‐bromo‐2‐nitropropane‐1,3‐diol and the acaricide dicofol were safe, while the insecticide phosalone and fungicide tridemorph were moderately toxic to adults. All other insecticides tested, namely dimethoate, fenitrothion, monocrotophos, phosphamidon, endosulfan, cypermethrin, decamethrin, fenvalerate and fluvalinate, and the fungicides carbendazim, methyl thiophenate and carboxin were toxic to adults. A high level of parasitism was recorded for all fungicide treatments and for dicofol and fluvalinate. The larval stage of the parasitoid was more tolerant than other stages. The residual toxicity of all fungicides, and dicofol, did not affect the ability of the parasitoid to parasitize its host, while the insecticides phosalone and fluvalinate were slightly persistent, favouring 44.7% and 49.3% parasitism after 15 days. Residues of dimethoate, decamethrin, cypermethrin, fenvalerate, monocrotophos and phosphanidon were moderately persistent, while fenitrothion and endosulfan were persistent.     

Mycolytic effect of fluorescent Pseudomonas in biocontrolling of fungal phytopathogenic Curvularia lunata,Fusarium oxysporum,Alternaria padwickii and Rhizoctonia solani

About 50 bacterial strains, each of Pseudomonas fluorescens, from different rhizospheric soil of different plants were screened for antagonistic activity against Curvularia lunata, Fusarium oxysporum, Alternaria padwickii, Rhizoctonia solani causing black kernel, kernel spotting, root rots, stackburn and sheath blight diseases of rice (Oryza sativa L.). Out of the 50 isolates, 15 isolates were found to be effective in lysing the cell wall of the above-mentioned putative pathogens tested in vitro. These Pseudomonas isolates produced mycolytic enzymes, viz. β-1,3-glucanases, β-1,4-glucanases and lipases. P. fluorescens PAK1 and PAK12 among the strains were more effective for the production of these enzymes while PAK12 produce good level of β-1,3-glucanases, β-1,4-glucanases and lipases against tested fungal pathogens. These findings demonstrate a mechanism of antagonism by P. fluorescens against different fungal plant pathogens.     

Enhancing biological control of basal stem rot disease (<Emphasis Type="Italic">Ganoderma boninense</Emphasis>) in oil palm plantations

Basal Stem Rot (BSR) disease caused by Ganoderma boninense is the most destructive disease in oil palm, especially in Indonesia and Malaysia. The available control measures for BSR disease such as cultural practices and mechanical and chemical treatment have not proved satisfactory due to the fact that Ganoderma has various resting stages such as melanised mycelium, basidiospores and pseudosclerotia. Alternative control measures to overcome the Ganoderma problem are focused on the use of biological control agents and planting resistant material. Present studies conducted at Indonesian Oil Palm Research Institute (IOPRI) are focused on enhancing the use of biological control agents for Ganoderma. These activities include screening biological agents from the oil palm rhizosphere in order to evaluate their effectiveness as biological agents in glasshouse and field trials, testing their antagonistic activities in large scale experiments and eradicating potential disease inoculum with biological agents. Several promising biological agents have been isolated, mainly Trichoderma harzianum, T. viride, Gliocladium viride, Pseudomonas fluorescens, and Bacillus sp. A glasshouse and field trial for Ganoderma control indicated that treatment with T. harzianum and G. viride was superior to Bacillus sp. A large scale trial showed that the disease incidence was lower in a field treated with biological agents than in untreated fields. In a short term programme, research activities at IOPRI are currently focusing on selecting fungi that can completely degrade plant material in order to eradicate inoculum. Digging holes around the palm bole and adding empty fruit bunches have been investigated as ways to stimulate biological agents.