Efficiency of Trichoderma harzianum as a biocontrol agent in combination with humate and chitosan against Meloidogyne incognita on tomato

Abstract

A pot trial was conducted to estimate the role of Trichoderma harzianum alone or in combination with two organic substances, potassium humate and chitosan in controlling Meloidogyne incognita on tomato. All treatments caused greater decreases in parameters of M. incognita in comparison to the control treatment (nematode only) and this led to noticeable enhancements in growth and yield of tomato. The lowest numbers of eggmasses, eggs/eggmass, galls/root, females/root, and second stage juveniles/250?g soil were recorded due to the combination of T. harzianum (10¹⁰ spore/ml) with chitosan and potassium humate after 120 days from the transplanting of tomato seedlings. Also, this treatment showed the best promotion for all tomato parameters (lengths and weights of shoots and roots, and productivity). So, mixing chitosan, potassium and T. harzianum is highly recommended to be used as an effective bio-nematicide against M. incognita on tomato plants.     

Enhanced tolerance of Mentha arvensis against Meloidogyne incognita (Kofoid and White) Chitwood through mutualistic endophytes and PGPRs

The use of herbs in pharmaceutical preparation is ever increasing, and the demand for pesticides free material by the concern industries is on the rise. Consequently the need to grow disease-free plants using non-chemical fertilizers and pesticides is the need of the hour. Mentha arvensis cv. kosi is highly infested with Meloidogyne incognita (Kofoid and White) Chitwood, and severe oil yield loss occurs due to this nematode pest. Employing ecofriendly ways of nematode management, the mutualistic endophytes (Trichoderma harzianum strain Thu, Glomus intraradices) and plant growth promoting rhizobacteria (Bacillus megaterium and Pseudomonas fluorescens) were assessed individually and in combination on plant biomass, oil yield of menthol mint (M. arvensis cv. kosi), reproduction potential and population development of root knot nematode, M. incognita under glasshouse conditions. These microbes enhanced the plant biomass and percent oil yield both with and without M. incognita inoculation. Dual application of mutualistic fungal endophytes and Plant Growth Promoting Rhizobacteria (PGPRs) may be a wise option for enhancing the oil yield and tolerance of menthol mint against M. incognita infection.     

Biological Control of Root-Knot Nematode <i>Meloidogyne incognita</i> in <i>Psoralea corylifolia</i> Plant by Enhancing the Biocontrol Efficacy of <i>Trichoderma harzianum</i> Using Press Mud

Meloidogyne incognita is a plant pathogen causing root-knot disease and loss of crop yield. The present study aimed to use Trichoderma harzianum as a biocontrol agent against plant-parasitic nematodes and used press mud, which is a solid waste by-product of sugarcane, as a biocontrol agent and biofertilizer. Therefore, the combined application of T. harzianum and press mud may enhance nematode control and plant growth. Elemental analysis of press mud using scanning electron microscopy (SEM) integrated with an Energy Dispersive X-ray (EDX) analyzer revealed the presence of different elements such as C, O, Mg, Si, P, K, Ca, Cu and Zn. In addition, a greenhouse study was conducted to investigate the combined effects of press mud and T. harzianum on M. incognita reproduction and growth and the biochemical features of Psoralea corylifolia. The results showed that plant length, dry biomass, leaf area, the number of seeds per plant, chlorophyll a, chl b, carotenoid content, nitrate reductase, carbonic anhydrase, and nitrogen content were significantly increased (P ≤ 0.05) in the T2 plants (plants were treated with 100 g of press mud + 50 mL T. harzianum before one week of M. incognita inoculation), over inoculated plants (IC). Antioxidant enzyme activity of ascorbate peroxidase (APX), catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) in the foliage of P. corylifolia was significantly increased when plants were treated with press mud + T. harzianum. A significant reduction in the number of egg masses, nematode population, and root-knot index (RKI) was found in plants with T2 plants. These results suggest that the combined application of T. harzianum and press mud has the potential to control the M. incognita infection and can be used as an environmentally safe alternative to chemical nematicides and also help in the removal of sugarcane waste that causes environmental pollution.     

In vitro studies of antagonistic fungi against the root-knot nematode,Meloidogyne incognita

The present study was carried out in vitro to determine the efficacy of indigenous fungi isolated from egg masses of root-knot nematode, Meloidogyne incognita on egg parasitism, egg hatching, mobility and mortality against root-knot nematode, M. incognita. The tested fungi were Acremonium strictum, Aspergillus terreus, A. nidulans, A. niger, Chetomium aubense, Chladosporium oxysporum, Fusarium chlamydosporium, F. dimarum, F. oxysporum, F. solani, Paecilomyces lilacinus, Pochonia chlamydosporia, Trichoderma viride and T. harzianum. All tested fungi showed varied effects against the nematodes. Culture filtrates of A. strictum was very effective against the nematode in regards to egg parasitism (53%), egg hatching inhibition (86%) and mortality (68%) compared to controls. A. strictum was found to have an advantage over P. lilacinus, P. chlamydosporia, T. viride and T. harzianum in that it caused greater mortality of the second stage juveniles (J₂). A. terreus did not show egg parasitism but was found to be highly toxic against second stage juveniles (J₂) causing high mortality (around 68%). Thus, A. strictum and A. terreus showed good biocontrol potential against root-knot nematode, M. incognita under in vitro conditions.     

In vitro evaluation of combination of Trichoderma harzianum and chitosan for the control of sapstain fungi

In vitro assays were undertaken to evaluate the control of two sapstain fungi, Leptographium procerum and Sphaeropsis sapinea by a combination of chitosan or chitosan oligomer and an albino strain of Trichoderma harzianum. Spore germination and hyphal growth of the test fungi were assessed on media amended with chitosan or chitosan oligomer with and without T. harzianum using either simultaneous inoculation with test fungus or inoculation 1, 2, or 3 days after pre-infection with test fungus.There was no mycelial growth of the test fungi regardless of chitosan concentrations used when either L. procerum or S. sapinea was simultaneously inoculated with T. harzianum. However, the dose–response of chitosan or chitosan oligomer on the test fungi was apparent when T. harzianum was not simultaneously inoculated with test fungus but introduced later. There was a greater growth reduction at higher concentrations (0.075–0.1% v/v) of chitosan, and overall chitosan oligomer was more effective than chitosan aqueous solution.Chitosan alone was able to restrict or delay the germination of spores but the combination of chitosan and T. harzianum inhibited spore germination and hence colony formation of test fungi regardless of time delay.     

An integrated approach to managing Rhizoctonia solani on maize in marginal soils: field studies

Abstract

A study was conducted over two seasons in a marginal soil with an acid saturation of 54%. A plant growth promoting rhizobacterium, Bacillus megaterium, a commercial biocontrol agent, Trichoderma harzianum Strain kd (Eco-T^®), and soluble potassium silicate were used in this study. The objective was to control maize yield reduction caused by Rhizoctonia solani AG2 root rot isolated from infected maize. R. solani reduced maize yields significantly by 34% in the first season. However, the losses decreased in the second season from 34% to 10%. In the first season, combination of T. harzianum, B. megaterium and potassium silicate increased maize yields by 130%. The highest yield in the presence of R. solani was treatments with T. harzianum (216%) followed by T. harzianum plus potassium silicate (214%) and lastly T. harzianum plus B. megaterium (178%). However, in the second season, T. harzianum plus potassium silicate treatment resulted in the highest yields.     

Enhanced growth and nutrition of micropropagated Ficus benjamina to Glomus mosseae co-inoculated with Trichoderma harzianum and Bacillus coagulans

A greenhouse investigation was conducted to study the influence of the arbuscular mycorrhizal (AM) fungus Glomus mosseae and the plant growth-promoting rhizomicroorganisms (PGPRs) Bacillus coagulans and Trichoderma harzianum on the growth and nutrition of micropropagated Ficus benjamina plantlets. The AM fungus was inoculated either singly or in combination with the PGPRs. Plants showed maximum plant height, biomass, P content, mycorrhizal root colonization, spore numbers and populations of T. harzianum and B. coagulans in root zone soil when all the three organisms were inoculated together. Thus, when G. mosseae co-inoculated with PGPRs enhances growth and nutrition of Ficus benjamina. T. harzianum and B. coagulans are thus designated as mycorrhizal helper organisms.     

Trichoderma Harzianum WKY1: an indole acetic acid producer for growth improvement and anthracnose disease control in sorghum

The efficiency of two isolates of Trichoderma harzianum (WKY1 and WKY5) as bio-control agents against anthracnose disease in sorghum was investigated. In vitro, T. harzianum WKY1 isolate showed superiority in terms of inhibition of both mycelial growth and spore germination of Colletotrichum sublineolum, the causative agent of sorghum anthracnose, as well as induction of the sorghum seed germination over T. harzianum WKY5 isolate. The culture filtrate of the selected isolate (T. harzianum WKY1) was analysed using GC-MS system to determine their chemical constituents. Twenty-nine components with varied existence percentages were identified. Although T. harzianum WKY1 produced the phytohormone indole-3-acetic acid (IAA) on tryptophan free medium, a marked dependency on tryptophan for the production of IAA was noticed. Nutritional components were optimized for maximizing IAA production using the central composite design. The optimum levels were 1.06, 29.86 and 2.93?g?L^?1 from tryptophan, sucrose and NaNO₃, respectively, with a maximum IAA biosynthesis (138.9?µg?mL^?1) after five days of incubation. Production of IAA in the culture filtrate of T. harzianum WKY1 was qualitatively and quantitatively analysed by LC-MS system using a reference standard of IAA. Under greenhouse conditions, application of T. harzianum WKY1 and/or its filtrate reduced greatly the disease severity as well as improved the plant growth of sorghum. From the present data, we can recommend the application of T. harzianum WKY1 as a dual purpose bio-agent for biological control of anthracnose disease and plant growth promotion.     

Integration of Glomus mosseae with Chromolaena odorata powder for suppression of Meloidogyne incognita on maize (Zea mays L.)

Meloidogyne incognita infestation on maize results in heavy yield loss in farmers’ field. Most of the varieties adopted by subsistence farmers in Nigeria are susceptible to M. incognita. Beside these, the cost of control exceeding the profit from the crop using nematicides and the pollution risk they pose to the environment has necessitated the need for alternatives. Pot and field experiments were, therefore, conducted to investigate the effects of Chromolaena odorata powder and Glomus mosseae (a mycorrhizal fungus) on M. incognita pathogenicity on maize. Hybrid Oba super II improved maize variety adopted by local farmers was selected for the study. Maize plants were grown with G. mosseae (5 spores/g of soil) and soil amended with C. odorata powder (1% w/w) singly and in combination. Two weeks after emergence, Test plants were inoculated with 5000 M. incognita eggs. Sixty days after planting, destructive samples were assessed for root gall symptom and severity, and nematode population. Results show that G. mosseae and C. odorata powder were effective in controlling the population of M. incognita and the root knot nematode symptom and gave the highest yield parameters in combination. Single application of G. mosseae and C. odorata powder was similar in the effect on M. incognita and maize yield. Combination of G. mosseae and C. odorata powder may become a viable alternative to nematicide in managing M. incognita pathogenicity on maize as C. odorata powder may serve as a carrier medium for G. mosseae.     

Management of <Emphasis Type="Italic">Sclerotium rolfsii</Emphasis> with integration of non-conventional chemicals,vermicompost and <Emphasis Type="Italic">Pseudomonas syringae</Emphasis>

Two non-conventional chemicals, ZnSO₄ (10⁻⁴ mM) and oxalic acid (4 mM) were tested (alone as well as in combination with seeds bacterized with Pseudomonas syringae strain PUR46 and vermicompost substitution in the potting soil), for their ability to suppress collar rot of chickpea (Cicer arietinum) caused by Sclerotium rolfsii under greenhouse conditions. ZnSO₄ and oxalic acid were applied as pre-inoculation foliar spray on chickpea and subsequently challenged with S. rolfsii. Both the chemicals provided significant protection to chickpea compared to control (100% plant mortality) when used alone as well as in combination with PUR46 and vermicompost. However, ZnSO₄ was more effective than oxalic acid against S. rolfsii. Amongst the treatments tried, plant mortality was least when ZnSO₄ was used in combination with seed bacterization with PUR46 and 25% vermicompost substitution. The findings indicate the utility of integration of the above factors in managing collar rot efficiently.     

Interactions of Ralstonia solanacearum and Pectobacterium carotovorum with Meloidogyne incognita on potato

Effect of interactions of Meloidogyne incognita with Ralstonia solanacearum and interaction of M. incognita with Pectobacterium carotovorum were studied in sequential and simultaneous inoculations on potato (Solanum tuberosum). Inoculation of M. incognita caused a lesser reduction in plant growth than caused by R. solanacearum. Inoculation of M. incognita plus R. solanacearum caused a greater reduction in plant growth than the damage caused by either pathogen. Inoculation of M. incognita prior to R. solanacearum resulted in a greater reduction in plant growth than R. solanacearum was inoculated prior to M. incognita. However, inoculation of M. incognita or P. carotovorum caused similar reduction in plant growth. Inoculation of P. carotovorum prior to M. incognita caused lesser reduction in plant growth than simultaneous inoculation of both pathogens. Inoculation of M. incognita caused galling in potato roots but the size of galls was small. Inoculation of P. carotovorum or R. solanacearum with M. incognita had adverse effect on galling and nematode multiplication. Wilting or soft rot index was 3 when R. solanacearum or P. carotovorum was inoculated alone. In other treatments, where R. solanacearum or P. carotovorum was inoculated with M. incognita, wilting or soft rot indices were 5.     

Integration of Pythium nunn and Trichoderma harzianum isolate T-95 for the biological control of Pythium damping-off of cucumber

Two biological control agents, Pythium nunn and Trichoderma harzianum isolate T-95, were combined to reduce Pythium damping-off of cucumber in greenhouse experiments lasting 3–4 weeks. T. harzianum T-95, a rhizosphere competent mutant, was applied to seeds and P. nunn was applied to pasteurized and raw soils naturally and artificially infested with Pythium ultimum. Some treatments were also amended with bean leaves to enhance the activity of P. nunn. The biological control of Pythium damping-off was evaluated in a Colorado soil (Nunn sandy loam) and an Oregon soil mix, which were replanted twice after 2 and 3 months. Interactions between P. nunn and T-95 were detected in the Colorado but not the Oregon soil. No consistent evidence of antagonism between P. nunn and T. harzianum was seen, and significant interactions were detected in the Colorado, but not the Oregon soil. In the first planting of some treatments, the combination of P. nunn and T. harzianum gave greater control of damping-off than either applied alone. P. nunn was most effective in soils that were pasteurized or amended with bean leaves. T. harzianum controlled Pythium damping-off in the Colorado, but not the Oregon soil. In both soils, disease declined over time in treatments amended with bean leaves but without P. nunn or T. harzianum added. This suppression was greater in the Colorado soil, which contained an indigenous population of P. nunn. This work demonstrates that two compatible biological control agents can be combined to give additional control of a soil-borne plant pathogen.     

Isolation and characterization of PRA1, a trypsin-like protease from the biocontrol agent <Emphasis Type="Italic">Trichoderma harzianum</Emphasis> CECT 2413 displaying nematicidal activity

Mycoparasitic Trichoderma strains secrete a complex set of hydrolytic enzymes under conditions related to antagonism. Several proteins with proteolytic activity were detected in culture filtrates from T. harzianum CECT 2413 grown in fungal cell walls or chitin and the protein responsible for the main activity (PRA1) was purified to homogeneity. The enzyme was monomeric, its estimated molecular mass was 28 kDa (SDS-PAGE), and its isoelectric point 4.7–4.9. The substrate specificity and inhibition profile of the enzyme correspond to a serine-protease with trypsin activity. Synthetic oligonucleotide primers based on N-terminal and internal sequences of the protein were designed to clone a full cDNA corresponding to PRA1. The protein sequence showed <43% identity to mammal trypsins and 47–57% to other fungal trypsin-like proteins described thus far. Northern analysis indicated that PRA1 is induced by conditions simulating antagonism, is subject to nitrogen and carbon derepression, and is affected by pH in the culture media. The number of hatched eggs of the root-knot nematode Meloidogyne incognita was significantly reduced after incubation with pure PRA1 preparations. This nematicidal effect was improved using fungal culture filtrates, suggesting that PRA1 has additive or synergistic effects with other proteins produced during the antagonistic activity of T. harzianum CECT 2413. A role for PRA1 in the protection of plants against pests and pathogens provided by T. harzianum CECT 2413 is proposed.     

Determination of the growth and solubilization capabilities of <Emphasis Type="Italic">Trichoderma harzianum</Emphasis> T1

The growth capability of Trichoderma harzianum Rifaii Tl was tested on Malt Extract and Czapeks Dox agar containing different concentrations of Cu²⁺, Zn²⁺, Mn²⁺, Fe²⁺ and Ca²⁺. The T. harzianum Tl isolate was observed to produce mycelia and spores in various mineral-containing media. It showed the lowest tolerance to Ca²⁺ and the highest tolerance to Fe²⁺. Solubilization capability of T. harzianum Tl for some insoluble minerals via acidification of medium has been tested on MnO₂, CuO, Fe₂O₃ and metallic Zn. T. harzianum Tl was able to solubilize MnO₂ and metallic Zn in a liquid medium.     

Use of Trichoderma harzianum cultured on ground mesocarp fibre of oil-palm as seed treatment to control Pythium aphanidermatum,a causal agent of damping-off of Chinese kale seedling

Trichoderma harzianum, isolate T 01-22, was cultured on either sorghum grains, ground mesocarp fibre of oil-palm or oil-palm shell, both amended with urea fertilizer (100:1, w/w). T. harzianum cultured on ground mesocarp fibre was then used to coat seeds of Chinese kale (Brassica alboglabra Bailey) to control damping-off of seedlings caused by Pythium aphanidermatum. Biomass of T. harzianum cultured on ground mesocarp fibre of oil-palm was more effective than Captan and Benomyl, but less effective than Metalaxyl, in controlling damping-off of Chinese kale seedlings. Viability of T. harzianum growing on sorghum grains was reduced significantly during 7 months storage, followed by that of T. harzianum cultured on ground mesocarp fibre and oil-palm shell, both amended with urea fertilizer (46-0-0) at 100:1 (w/w).     

Suppression of sugar beet damping-off caused by Rhizoctonia solani using bacterial and fungal antagonists

Rhizoctonia damping-off caused by Rhizoctonia solani Kühn, is one of the most damaging sugar beet diseases. It causes serious economic damage wherever sugar beets are grown. Biological control is an efficient and environmentally friendly way to prevent damping-off disease. Suppression of damping-off disease caused by R. solani was carried out by four isolates of Bacillus subtilis (Ehrenberg) Cohn as well as three isolates of each of Trichoderma harzianum Rifai and Trichoderma hamatum (Bonord.) Bainier. The effect of Bacillus and Trichoderma isolates against R. solani was investigated in vitro and tested on sugar beet plants under greenhouse conditions. Isolates of Bacillus and Trichoderma were able to inhibit the growth of R. solani in dual culture. Furthermore, Trichoderma isolates gave high antagonistic effect than isolates of B. subtilis. Under greenhouse conditions, coating seeds by T. harzianum and B. subtilis separately, reduced seedling damping-off significantly. However, applications of T. harzianum increased the percentage of surviving plants more than B. subtilis in comparison to control. The obtained results indicate that T. harzianum and B. subtilis are very effective biocontrol agents that offer potential benefit in sugar beet damping-off and should be harnessed for further biocontrol applications.     

Effect of organic fertiliser and Chromolaena odorata residue on the pathogenicity of Meloidogyne incognita on maize

Pot experiments laid out in a complete randomised design were conducted in the screen house of the Department of Crop Protection, University of Agriculture, Abeokuta, Ogun State, Nigeria to determine the effects of organic fertiliser and Chromolaena odorata residue at 1% w/w on the pathogenicity of Meloidogyne incognita infecting maize. M. incognita significantly reduced the plant height, number of leaves per plant, leaf area, cob weight and grain yield of maize by 6.89, 15.18, 20, 63.92 and 56.16% respectively. C. odorata residue and organic fertiliser significantly suppressed M. incognita galling, inhibited the nematode fecundity and reduced the number of eggs and juveniles on maize. A remarkable increase in plant height, number of leaves per plant, leaf area, cob weight and grain yield were observed on maize plants treated with the mixture of C. odorata and organic fertiliser despite the nematode infection. The observation from this study suggests that C. odorata in combination with organic fertiliser is a viable option for the control of M. incognita on maize.     

Specific PCR assays for the detection of <Emphasis Type="Italic">Trichoderma harzianum</Emphasis> causing green mold disease during mushroom cultivation

We have developed a polymerase chain reaction (PCR)-based detection method for Trichoderma harzianum, which causes green mold disease in mushroom cultivation fields and facilities. Based on the sequence data of the internal transcribed spacer (ITS) region of T. harzianum strains and several other species, six primers consisting of three forward and three reverse primers were designed. Among the nine possible combinations of these primers, PCR with the pair THITS-F2 and THITS-R3 distinguished most T. harzianum strains from other Trichoderma species. The optimal annealing temperature for detection of T. harzianum strains was from 62° to 63°C with this primer combination. We designed new primers derived from THITS-F2 and THITS-R3. Annealing temperatures to detect T. harzianum ranged from 64° to 67°C using the new primers. The detection limit of T. harzianum DNA was 50 fg by nested PCR with THITS-F1 and LR1-1 for the first PCR and the new primers for the second PCR. T. harzianum was readily detectable in contaminated cultures of Lentinula edodes by this method.     

Effect of chitosan on physiological, morphological, and ultrastructural characteristics of wood-degrading fungi

An investigation was undertaken to understand the mechanism(s) by which chitosan exerts its antifungal effects against the wood-degrading fungi Sphaeropsis sapinea and Trichoderma harzianum. Exposure to increasing concentrations of chitosan caused an increase in the amount of hydrogen peroxide accumulation in cultures of S. sapinea, which was accompanied by a decrease in superoxide formation. The same effect was not observed in T. harzianum. Potassium ion leakage was an early event for both test fungi, leakage being more pronounced for S. sapinea than T. harzianum for the first 5 min, particularly at higher concentrations of chitosan treatment. Fluorescence microscopy provided evidence that the effect of chitosan on fungal hyphae was mediated through alterations in the plasma membrane properties. Chitosan also severely affected fungal morphology. Increasing concentrations of chitosan induced excessive branching, vacuolation, and a reduction in hyphal diameter. Transmission electron microscopy, which showed more severe ultrastructural changes in S. sapinea hyphae from chitosan treatment as compared to T. harzianum, provided valuable complementary information. The data suggest that the plasma membrane may be the primary target of chitosan action, and that the two fungi differ in the extent to which they are affected.     

Combining the oomycete Pythium oligandrum with two other antagonistic fungi: Root relationships and tomato grey mold biocontrol

To reduce Pythium oligandrum biocontrol variability and improve its efficacy, experiments were performed by combining the oomycete with two other antagonistic fungi, Fusarium oxysporum strain Fo47 and Trichoderma harzianum. In Petri dishes, Fo47 or T. harzianum hyphae destroyed P. oligandrum cells by antibiosis and mycoparasitism processes; in the rhizosphere of tomato plants (Lycopersicon esculentum), the same antagonistic features were observed. However, in the rhizosphere, hyphae are frequently separated by a certain distance; this allows the coexistence and the persistence of the three microorganisms on the root systems. When introduced in the rhizosphere, Fo47 and P. oligandrum were able to penetrate the root tissues with Fo47 limited to the epidermal and upper layers of cortical cells while P. oligandrum colonized deeper tissue at a faster rate. The two antagonists were killed in few days within roots following elicited plant-defense reactions. T. harzianum was not able to penetrate root tissues. Root colonization with either P. oligandrum alone or in combination with Fo47 and/or T. harzianum resulted in systemic plant resistance which provided plant protection against Botrytis cinerea infection of leaves. The level of control and the expression of pathogenesis-related proteins (PR-proteins) in leaves were similar whatever the antagonistic microbial treatment applied to roots.