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).     

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.     

Antagonistic actions of Pythium oligandrum and Trichoderma harzianum against phytopathogenic fungi (Fusarium oxysporum and Pythium ultimum var. ultimum)

Abstract

The possible biological control of damping-off fungi, Fusarium oxysporum and Pythium ultimum by Pythium oligandrum or Trichoderma harzianum was in vitro investigated. Results of comparing the antagonistic activity of P. oligandrum and T. harzianum in dual plates against the tested phytopathogens indicated different degrees of antagonism. After 12 days of incubation colony of the phytopathogenic fungus was completely overgrown by the antagonist, except for the interaction between T. harzianum and F. oxysporum which showed no overgrowth or any hyphal penetration by the antagonist. However, growth and proliferation of F. oxysporum colony was repressed. T. harzianum and P. oligandrum produced chitinase and β-1,3-glucanase when they were grown on liquid culture medium supplemented with chitin or fungal dried mycelium as a sole carbon source, and enzyme production was higher by T. harzianum comparing with P. oligandrum under the same condition. Fungal dried mycelium of F. oxysporum was the most selective carbon source for enzyme production, on the other hand, chitinase production was significant locked when P. ultimum dried mycelium was used as a carbon source. Production of volatile compounds by P. oligandrum or T. harzianum against F. oxysporum and P. ultimum was examined using the inverted plates method. F. oxysporum was inhibited by the antagonist volatile compounds and it is inhibited 100% by increasing the amount of inoculum size. Production of potential biocontrol agents provided with economically features and working under field conditions are recommended.     

Ecology of Hymexazol-Insensitive Pythium Species in Field Soils

Soils from 100 irrigated fields (95 under vegetables, 5 under citrus) in different geographical locations in the West Bank (Palestinian Autonomous Territory) were surveyed for hymexazol-insensitive (HIS) Pythium species using the surface soil dilution plate (SSDP) method with the VP3 medium amended with 50 mg/L hymexazol (HMI) (VP3H50), over a period of 12 months. HIS Pythium species were isolated from 37% of the soils surveyed, with mean population levels ranging from 4.3-1422 CFU g(-1) dry weight. Eight HIS Pythium taxa were recovered on the VP3H50 medium, the most abundant of which was P. vexans (found in 29% of field soils surveyed). Seasonal variations in population levels of HIS Pythium species were studied in four fields over a period of 12 months. Significant seasonal variations in HIS population levels were detected in the four fields, with the highest population levels of HIS Pythium spp. encountered in spring and the lowest population levels in winter in three of the fields surveyed. Effects of HMI on linear growth and colony morphology of 149 Pythium ssp. isolates were examined on CMA amended with HMI at five concentrations. Pythium vexans isolates responded differently from those of the other Pythium species. Isolates of this important pathogen were more insensitive to HMI at high concentrations than the other main species tested. A large proportion of the P. ultimum isolates was either insensitive or weakly sensitive to HMI. Furthermore, a few isolates of other Pythium species were insensitive to the fungicide at various concentrations. The colony morphology of P. vexans isolates was not affected by HMI, whereas colonies of the other species showed sparse growth on the HMI amended medium relative to the control. The pathogenicity of P. vexans and P. ultimum isolates to cucumber seedlings was examined in growth chambers. Insensitive isolates of both species were found to be more virulent damping-off pathogens than the sensitive isolates. The present study demonstrates that HMI can not be used effectively in controlling Pythium spp. in soil inhabited with high densities of HIS Pythium spp. pathogens.     

Prof. Dr. Lothar Behr

A damping-off disease of wheat was shown in a wheat field in Kidwan village, El-Minia city, Egypt, during December, 2000. Pythium diclinum was the causal agent of such disease and this is the first reported work of its isolation as a disease to wheat. Wheat seedlings collected from that field showed browning lesions at the basal part and wilting followed by damping-off. Examination of root pieces and other infected parts yielded only Pythium diclinum. The pathogen was characterized by its typically filamentous zoosporangia, diclinous antheridia and aplerotic thick-walled oospores. Pathogenicity of this fungus was determined on wheat under greenhouse conditions and P. diclinum was proved to be pathogenic on wheat. Two isolates of each of Gliocladium roseum and Trichoderma harzianum were tested for their bio-control activity against damping-off disease of wheat caused by P. diclinum. Incorporation of G. roseum or T. harzianum isolates into carboxymethylcellulose seed coating successfully eliminated pre-emergence damping-off of the wheat caused disease, whereas post-emergence damping-off was prevented by adding inocula of each of the two fungi separately to the infested soil with P. diclinum.     

Mycotic flora of the interdigital spaces of the human foot: A preliminary investigation

Culture filtrates of Trichoderma harzianum Rifai have been found to inhibit zoospore germination, germ tube elongation and mycelial growth of Pythium aphanidermatum causing the damping-off disease of tobacco in Nigeria. Further, the invasion of the hyphae of P. aphanidermatum by hyphae of T. harzianum has been demonstrated in mixed cultures of the two fungi. The results of investigations on the role of T. harzianum as an agent of biological control of the damping-off disease in sterilized and unsterilized soils are reported and discussed.     

Use of Trichoderma harzianum and Gliocladium vitens for the Biological Control of Post-emergence Damping-off and Root Rot of Cucumbers Caused by Pythium ultimum

The antagonist strains Gliocladium virens G2 and Trichoderma harzianum T3 originally isolated from Pythium suppressive peat, and two benomyl-resistant strains of T. harzianum, T12B and T95, were evaluated as biological control agents of damping-off and root rot of cucumbers in sphagnum peat caused by Pythium ultimum. All strains were equally effective when applied as 1 % peat-bran preparations, whereas the effectiveness of disease control was reduced at higher concentrations of the antagonists. The two wild-type strains were also found to be effective when applied as conidial suspensions, and in this case no reduction in disease control was seen at higher concentrations. G. virens G2 and T. harzianum T12B showed antibiotic activity against P. ultimum in in vitro tests; however there were no signs of mycoparasitism of P. ultimum by any of the antagonist strains.     

Epicoccum Nigrum as biocontrol agent of Pythium damping-off and root-rot of cotton seedlings

Efficacy of Epicoccum nigrum and its exudate was tested in control the pre- and post-emergence damping-off and root-rot of Egyptian cotton (cv. Giza 83) in vitro and in vivo. Different isolates of Epicoccum nigrum reduced the radial growth of both Pythium debaryanum and P. ultimum significantly, by production considerable inhibition zones. In liquid cultures E. nigrum exudate showed a high fungicidal effect resulting in a significant reduction of the mycelial dry weight of the two investigated Pythium spp. Also E. nigrum exudate inhibited cellulase and pectinase activity by P. debaryanum and P. ultimum. Soaking of cotton seeds in E. nigrum exudate for different intervals resulted in significant reduction of root-rot severity of seedlings as well as the contamination of seeds and seedlings by fungi during and after germination. These treatments also stimulated germination of cotton seeds and enhanced the seedlings vigour significantly. In pot experiments, the use of E. nigrum as a soil mixture or seed dressing significantly alleviated the hazard effect of P. debaryanum. Pythium ultimum seemed to be weak or non pathogen to the used cotton cultivar (Giza 83). Application of E. nigrum or its exudate not only involved in protection of cotton seedlings against Pythium damping-off and root-rot but also enhanced their vigour and growth characteristics. The main conclusion of this study is that E. nigrum could be used successfully as environmentally safe and economic biological control agent to protect cotton (cv. Giza 83) from damping-off and root-rot diseases caused by P. debaryanum.     

First records of Pythium aquatile and P. macrosporum isolated from soils in Japan

Pythium aquatile and P. macrosporum were isolated from the soil of a cultivated field in Gunma Prefecture and a forest in Nagano Prefecture for the first time in Japan. Their morphological characteristics are described, and their pathogenicity and taxonomy are discussed.     

Antagonistic Activities of Trichoderma harzianum Against Pythium aphanidermatum and Pythium myriotylum on Tobacco

Trichoderma harzianum rendered Pythium aphanidermatum and P. myriotylum non-viable in Petri dish dual culture. The Pythium mycelia from such cultures showed natural autofluorescence in the regions of interactions, indicating their death. Non-volatile and volatile fungicidal activities were detected in T. harzianum culture. Lytic activity of β-(l,3)-glucanase was detected on the cell walls of the Pythium spp. There was a significant decrease in the disease incidence when T. harzianum was incorporated into sterile soil, whereas the effect was insignificant in natural soil.     

Interaction of the mycoparasite Pythium oligandrum with other Pythium species

Interactions of Pythium oligandrum and four plant‐pathogenic Pythium spp. (P. ultimum, P. vexans, P. graminicola and P. aphanidermatum,) were studied in vitro by (i) video microscopy of hyphal interactions on water agar films, (ii) counting of host and mycoparasite propagules in different regions of opposing colonies on sunflower‐seed extract agar films and (Hi) ability of P. oligandrum to overgrow plates of potato‐dextrose agar previously colonized by Pythium spp. Pythium oligandrum typically coiled round the hyphae of Pythium hosts and penetrated the host hyphae after approximately 50 min from the hyphal coils, causing disruption of host hyphal tips up to 1.2 mm ahead of contact points. The relative growth rates of mycoparasite and host hyphae, timing of penetration and distance (sub‐apical) at which penetration led to host tip disruption were used to assess the potential of mycoparasitism by P. oligandrum to prevent the growth of Pythium hosts. P. aphanidermatum was unique among the ‘host’ Pythium spp. in being largely unaffected by P. oligandrum and in antagonizing the mycoparasite by coiling and penetrating the mycoparasite hyphae. Other host Pythium spp. apparently differed in susceptibility, the most susceptible being P. vexans and P. ultimum, whereas P. graminicola was more resistant. The results are discussed in relation to the role of P. oligandrum as a biocontrol agent, especially for limiting the ability of other Pythium spp. to increase their propagule populations in crop residues.     

Long-term changes in frequencies ofPythium iwayamai andP. paddicum in upland- and paddy-field soils

The long-term changes in the frequencies ofPythium iwayamai andP. paddicum in upland- and flooded paddy-field soils were compared. The frequencies of both species fell in both soils within the first 6 months after they had been buried. After 36 and 48 months,P. iwayamai showed higher frequency thanP. Paddicum in upland-field soil, whileP. paddicum showed higher frequency thanP. iwayamai in paddy-field soil. These findings indicate respectively higher colonization ability to organic matters ofP. iwayamai in upland-field soil andP. paddicum in paddy-field soil.This study was partly supported by a Grant-in-Aid (no. 63560048) from the Ministry of Education, Science and Culture, Japan.     

<Emphasis Type="Italic">Penicillium frequentans</Emphasis> isolated from<Emphasis Type="Italic"> Picea glehnii</Emphasis> seedling roots as a possible biological control agent against damping-off

Picea glehnii seedlings are affected by damping-off fungi in nurseries. The aims of this study were (1) to isolate fungi grown in the seedling rhizosphere in forest soil of P. glehnii, (2) to select fungi that produce antifungal compounds against Pythium vexans, and (3) to examine whether or not selected fungi can protect seedlings from P. vexans. Penicillium frequentans from Picea glehnii seedling roots produced antibiotic penicillic acid. Penicillic acid did not cause significant phytotoxicity to the seedlings. Penicillium frequentans increased the average percentage of surviving seedlings when inoculated together with Pythium vexans, but the increase was not significant. Vigorous mycelial growth of P. frequentans around seedling roots seems to be one of the mechanisms for protection, but the amount of penicillic acid was too low to show antifungal activity in the seedling rhizosphere.     

Effect of Trichoderma harzianum on microelement concentrations and increased growth of cucumber plants

The potential of the biocontrol agent Trichoderma harzianum strain T-203 to induce a growth response in cucumber plants was studied in soil and under axenic hydroponic growth conditions. When soil was amended with T. harzianum propagules, a 30% increase in seedling emergence was observed up to 8 days after sowing. On day 28, these plants exhibited a 95 and 75% increase in root area and cumulative root length, respectively, and a significant increase in dry weight (80%), shoot length (45%) and leaf area (80%). Similarly, an increase of 90 and 30% in P and Fe concentration respectively, was observed in T. harzianum inoculated plants. To better characterize the effect of T. harzianum during the early stages of root colonization, experiments were carried out in a gnotobiotic hydroponic system. An increased growth response was apparent as early as 5 days post-inoculation with T. harzianum, resulting in an increase of 25 and 40% in the dry weight of roots and shoots, respectively. Similarly a significant increase in the concentration of Cu, P, Fe, Zn, Mn and Na was observed in inoculated roots. In the shoots of these plants, the concentration of Zn, P and Mn increased by 25, 30 and 70%, respectively. Using the axenic hydroponic system, we showed that the improvement of plant nutritional level may be directly related to a general beneficial growth effect of the root system following T. harzianum inoculation. This phenomenon was evident from 5 days post-inoculation throughout the rest of the growth period, resulting in biomass accumulation in both roots and shoots.     

Chemical,physical and biological control of carrot seedling diseases

In naturally infested soil containingPythium ultimum, P. acanthicum andPhytophthora megasperma, onlyP. ultimum was associated with root rot and damped-off seedlings. Damping-off was promoted by low soil temperatures and by flooding. Seedling stands were markedly reduced when seed was pre-incubated in soil at 12°C but not at 25°C or 35°C. Dusting carrot seed with metalaxyl significantly increased seedling stands in the field at rates from 1.5–6 g kg⁻¹ seed and in both flooded and unflooded, naturally infested soil at 3.15 g kg⁻¹. In greenhouse experiments using artifically infested soil,P. ultimum andP. paroecandrum caused damping-off of carrot seedlings andRhizoctonia solani reduced root and shoot weights.R. solani caused damping-off in nutrient-enriched soil.P. acanthicum andP. megasperma were not pathogenic to seedlings, although both fungi colonized roots. Soil populations of allPythium spp., particularlyP. ultimum, increased during growth of seedlings and population growth ofP. megasperma was promoted by periodic flooding. Infestation of soil withP. acanthicum did not reduce damping-off of carrot seedlings byP. ultimum orP. paroecandrum, but significantly increased root and shoot weights and decreased root colonization byR. solani P. acanthicum has potential as a biocontrol agent againstR. solani.     

Impact of biocontrol agents in combination with Prosopis juliflora (Swartz) DC. in controlling the root-infecting fungi of leguminous crops

Biocontrol agents, viz., Rhizobium meliloti, Pseudomonas aeruginosa, Aspergillus niger and Trichoderma harzianum, are used as seed dressing and soil is amended with Prsosopis juliflora (Swartz) DC. plant parts like stem, leaves and flower at 1% w/w for the control of root-rot fungi. All antagonists suppressed the infection of root-rot fungi viz., Fusarium spp., Rhizoctonia solani and Macrophomina phaseolina whereas the infection of R. solani and M. phaseolina was controlled when cowpea (Vigna unguiculata L.) and mungbean (Vigna radiata L.) seeds were treated with P. aeruginosa and T. harzianum and the soil was amended with P. juliflora leaves’ powder at 1% w/w. However, germination of both the crops was observed in all treatments. Growth parameters like shoot and root length, shoot and root weight, and leaf area significantly increased in all the treatments as compared to the control parameters. P. aeruginosa and T. harzianum in combination with soil amendment with P. juliflora plant parts at 1% w/w were the most effective for the control of root-rot fungi of leguminous plants.     

Isolation of Pythium oligandrum from Egyptian soil and its mycoparasitic effect on Pythium ultimum var. ultimum the damping-off organism of wheat

Pythium oligandrum Drechsler bearing spherical sporangia with complex subglobose elements was isolated for the first time in Egypt from agricultural field soil cultivated with alfalfa (Trifolium alexandrinum) in El-Minia, Egypt. This fungus was found to be an active bio-control agent against P. ultimum var. ultimum, the damping-off organism of wheat. In agar plates, P. oligandrum parasitized P. ultimum var. ultimum hyphae with the aid of thin haustorial branches or infection pegs, eventually leading to host destruction. Incorporation of P. oligandrum into carboxymethylcellulose seed coating successfully eliminated pre-emergence damping-off of wheat caused by P. ultimum var. ultimum, whereas Post-emergence damping-off was prevented by adding inocula of P. oligandrum to the soil. This revised version was published online in June 2006 with corrections to the Cover Date.     

Isolation of Pythium Acanthicum,P. oligandrum,and P. periplocum from Soil and Evaluation of Their Mycoparasitic Activity and Biocontrol Efficacy Against Selected Phytopathogenic Pythium Species

Mycoparasitic Pythium species with spiny oogonia were surveyed in 50 Palestinian agricultural fields subject to different cropping practices using the Sclerotia Bait Technique (SBT) and the Surface-Soil-Dilution-Plate method (SSDP) with the selective VP3 medium. The mycoparasitic Pythium species were obtained from 21 (42%) soils using the SSDP method and from 37 (74%) soils using SBT. Pythium acanthicum and P. oligandrum were isolated by both methods, whereas P. periplocum was isolated only by the SBT. Using a newly modified dual plate culture method (MDPCM), the three mycoparasites showed varying antagonistic performance against several Pythium host species under a range of in vitro conditions. However, P. periplocum and P. oligandrum were found to be active biocontrol agents against P. ultimum, the damping-off organism of cucumber. This pathogen was antagonized, on thin films of water agar, by the three mycoparasites, and was moderately susceptible to P. periplocum while slightly susceptible to P. acanthicum and P. oligandrum. In direct application method in which antagonistic mycoparasites were incorporated into peat/sand mixture artificially infested with P. ultimum under growthroom conditions, Pythium oligandrum and P. periplocum (at 500 CFUg⁻¹) significantly improved seedling emergence and protected seedlings from damping-off. In the seed coating method, biocontrol by two types of seed dressing (homogenate- or oospore coated seeds), was comparable to that achieved by direct application. This revised version was published online in June 2006 with corrections to the Cover Date.     

Use of Oospore Formulations of Pythium oligandrum for Biological Control of Pythium Damping-off in Cress

Oospore preparations of Pythium oligandrum produced by liquid and solid-substrate fermentations were evaluated for biocontrol activity against Pythium damping-off in cress in artificially infested sand and naturally infested soil. Oospore biomass preparations from liquid fermentation of six isolates of P. oligandrum were equally effective in reducing damping-off in sand when tested as seed-coatings, whereas this type of preparation of a single isolate formulated as a kaolin dust, on Perlite and as alginate pellets incorporated into sand gave little or no control. None of the formulations containing oospores produced by solid-substrate fermentation incorporated into sand had any effect. In soil, a formulation containing oospores produced in a barley-Perlite solid-substrate fermentation and all oospore-biomass formulations which were prepared increased seedling survival, but none of these were as effective as a propamocarb HCl drench.     

Biocontrol of <Emphasis Type="Italic">Pythium</Emphasis> damping-off in cucumber by arbuscular mycorrhiza-associated bacteria from the genus <Emphasis Type="Italic">Paenibacillus</Emphasis>

The Pythium biocontrol features of 17 Paenibacillus strains, all previously isolated from the rhizosphere, hyphosphere or bulk soil from mycorrhizal and non-mycorrhizal cucumber plants, were examined using a cucumber seedling emergence bioassay. Thirteen strains – four strains of Paenibacillus polymyxa, eight strains of P. macerans and one strain of Paenibacillus sp. – significantly increased the percentage of seedling emergence of seeds inoculated with agar plugs of Pythium aphanidermatum FC42. Overall, the efficacy of Pythium biocontrol did not seem to differ between isolates of Paenibacillus originating from either mycorrhizal or non-mycorrhizal systems. No strains significantly reduced the damping-off incidence caused by the aggressive isolate Pythium sp. B5. Two strains of P. macerans not only reduced the incidence of pre-emergence damping-off by 73%, but they also counteracted the plant growth-depressing effect of P. aphanidermatum FC42, so that 68–82% of the emerged seedlings remained healthy 7 days after sowing. Two strains of P. macerans and one strain of P. polymyxa also significantly increased the percentage of seedling emergence following inoculation with approximately 10⁵ zoospores of P. aphanidermatum FC42. There was no significant difference between the dry weight of three selected bacteria-inoculated and -uninoculated plants in the absence of Pythium; however, the dry weight of bacteria-inoculated plants was significantly higher than that of the uninoculated control plants with bacteria in the presence of P. aphanidermatum FC42.