The significance of ecology in the development of Verticillium chlamydosporium as a biological control agent against root-knot nematodes (Meloidogyne spp.)

A thorough understanding of the interactions which occur between nematode parasites and nematode pests and the influence of biotic and abiotic factors on these interactions, is essential in the development of biological control agents for nematodes. The aim of this study was to develop a particular isolate of the nematophagous fungus Verticillium chlamydosporium as a biological control agent for root-knot nematodes. The work has gained insight into some of the key factors which govern the efficacy of the fungus as a biological control agent. The development of a semi-selective medium for V. chlamydosporium made it possible to study the growth, survival and spread of this fungus in non-sterile soil and on different parts of the root system as affected by soil-type, fungal density, nematode density, nematode species, temperature and watering.The V. chlamydosporium isolateThe isolate of V. chlamydosporium used in these studies was effective against Meloidogyne incognita, M. javanica , M. arenaria and M. hapla , and had no plant pathogenic characteristics. The fungus can be regarded as a typical egg-parasite, a characteristic which made it unsuitable for preventing initial nematode damage. However, effective population control in excess of 80% gave significant damage control after more than one nematode generation. After one generation of nematodes, population control achieved with the fungus was comparable with a nematicide treatment of aldicarb equivalent to 3.75 kg a.i./ha.The importance of ecological factors on the efficacy of V. chlamydosporiumThe efficacy of V. chlamydosporium as a biological control agent against rootknot nematodes is governed by four key factors:(a) Fungal establishment on the rhizoplane.(b) The proportion of egg-masses of Meloidogyne spp. exposed on the rhizoplane.(c) Rate of fungal growth relative to nematode development.(d) Spread of the fungus through soil.Fungal establishment on the rhizoplane : This is dependent on:1. Soil type . Mineral soils support less fungal proliferation than organic ones. This results in a slower build up of fungus on the rhizoplane when plants are grown in mineral soil compared with plants grown in organic soil.2. Application rate . This determines the initial fungal establishment on the rhizoplane. Presumably, because there are more nutrients available for the growth of a small initial fungal inoculum on the rhizoplane than for a large one, initial differences in fungal establishment on the roots tend to disappear after several weeks.3. Nematodedensity/gailing . Root galls induced by Meloidogyne spp. stimulate fungal growth on the gall surface.4. The host plant . Verticillium chlamydosporium differs in its ability to colonise the rhizoplane of different host plants, with for example roots from tomato, maize, cabbage and potato plants being readily colonised by the fungus, while roots from pepper, sorghum, soy-bean, pigeon pea and cotton plants are far less readily colonised.Exposure of egg-masses of Meloidogyne on the rhizoplane . This depends on:1 . Nematode density . Galling on tomato plants is much more severe when there is a high density of nematodes feeding in the roots, resulting in a proportion of of egg-masses staying embedded in the gall tissue at high nematode densities. These embedded egg-masses are physically protected from fungal attack.2. Temperature . Low temperatures (around 20°C) result in the induction of larger galls compared with those produced at higher temperatures (25°C-30°C). At 20°C, the egg-masses produced by M. incognita and M. javanica are in generalsmaller than those produced between 25°C and 30°C. The combination of these effects results in fewer egg-masses being exposed at lower temperatures.3. Nematode species . Galls induced by M. arenaria are larger than those induced by M. incognita or M. javanica . Depending on temperature this means that more egg-masses might stay embedded in gall-tissue when roots are infested by M. arenaria than with infestations of M. incognita or M. javanica .4. Host plant . There are marked differences in gall-size between different host plants infested with Meloidogyne. This undoubtedly has consequences for the proportion of egg-masses being exposed on the root-surface. However, data on this subject are not presented in this thesis.Rate of fungal growth relative to nematode development . This depends on:1 . Temperature . At temperatures below 25°C V. chlamydosporium is able to infect eggs before they mature and contain second-stage juveniles. At temperatures above 25°C nematode eggs develop faster than the fungus can infect them. This results in a proportion of eggs developing into fully embryonated eggs and juveniles at those temperatures. Juveniles and fully embryonated eggs are far less susceptible to fungal infections and it can be assumed that they have escaped further fungal attack. At 30°C. this may result in more than 40% of eggs escaping fungal infection.2. Aeration . Aeration influences the rate with which nematodes develop as well as the extend of fungal growth. It seems that, when oxygen is in short supply, the fungal growth rate is slower than nematode development. This factor however needs further investigation.Spread through soil . Numbers of colony forming units (cfu) can increase rapidly in soil when V. chlamydosporium is introduced as a chlamydospore inoculum into non sterile soil. Increase in numbers of cfu in soil is related to the soil temperature, and is most likely explained by the formation of conidia. These conidia can be moved by water percolating through soil. Roots of tomato plants became extensively colonised in this way up to 20 cm away from the original inoculum source, nine weeks after inoculation. This resulted in 100% of exposed egg-masses being colonised by the fungus. However, spread of conidia is dependent on:1 Watering . When the top 10 cm of the soil was inoculated with fungus, conidia spread more rapidly and more evenly when water was added from the top in comparison with watering from below.2. Water filled pores . When surplus water was allowed to drain out of the water saturated soil, subsequent waterings had no significant effect on further movement of conidia.3. Soil type . This factor needs further investigation, but it is likely that movement of conidia is greater in soils with a coarse soil texture and large pores than in soils with a fine soil texture and narrow pores.The practical implications of the studyThe ecological factors which govern the efficacy of V. chlamydosporium as a biological control agent against Meloidogyne were studied in the glasshouse. It was encouraging that introduction of V. chlamydosporium into field soil in a micro-plot test resulted in more than 90% population reductions of M. hapla on tomatoes, showing that data obtained from pot-tests were relevant in the field. The nematicide aldicarb (application rate: 3.75 kg a.i./ha) applied in combination with the fungus had no detrimental effects on fungal establishment in soil or on the root-surface. Fungal efficacy was therefore not influenced by the nematicide, resulting in greater control (98%) when both control measures were combined.The ecological studies presented in this thesis have practical implications for the use of V. chlamydosporium in the control of root-knot nematodes. The insights into the ecology of the soil might also be useful in the development of cultural practices to enhance soil suppressiveness in field soils

Produção de clamidósporos de Pochonia chlamydosporia em diferentes substratos

Clamidósporos são estruturas de sobrevivência do fungo nematófago Pochonia chlamydosporia. Objetivou-se, com este trabalho, avaliar diferentes substratos, teores de água e formas de inóculo para a produção in vitro de clamidósporos de P. chlamydosporia. Inicialmente, testaram-se os substratos grãos de milho triturado, grãos de arroz e casca de café e os tipos de inóculo meio líquido concentrado ou diluído (1:40) e discos de cultura, colonizados por P. chlamydosporia. Posteriormente, testou-se o substrato grãos de milho triturado suplementado ou não com caldo de batata com os inóculos do fungo nas formas de discos de micélio ou suspensão aquosa. As maiores produções de clamidósporos g-1 de substrato foram obtidas nos substratos grãos de milho triturado e casca de café, e as melhores formas de inóculo foram meio líquido diluído (1:40) e discos de micélio. A suplementação do substrato grãos de milho triturado com caldo de batata não aumentou a produção de clamidósporos do fungo e a melhor forma de inóculo do fungo foi a de discos de micélio. Além disso, testaram-se os substratos canjica, grãos de arroz, casca de café e fibra de coco, umedecidos com diferentes quantidades de água; o substrato casca de café umedecido por diferentes períodos; a casca de café enriquecida ou não com sacarose e farinha de arroz e o substrato grãos de arroz esterilizado no forno micro-ondas com diferentes quantidades de água. Apenas o substrato grãos de arroz, em todas as quantidades de água testadas, apresentou maior produção de clamidósporos. Não houve diferença na produção de clamidósporos no substrato casca de café umedecido por diferentes períodos e apenas quando a casca de café foi enriquecida com farinha de arroz, apresentou maior média de clamidósporos g-1 de substrato. Todos os tratamentos apresentaram grande produção de clamidósporos g-1 quando o substrato utilizado foi o de grãos de arroz tratados no forno de micro-ondas. O melhor meio de cultivo de P. chlamydosporia para a produção de clamidósporos foi o substrato contendo grãos de arroz.Chlamydospores are survival structures of the nematophagous fungus Pochonia chlamydosporia. The objective of this study was to evaluate different substrates, different contents of water and kinds of inoculum for the production of Pochonia chlamydosporia chlamydospores. The substrates evaluated were: milled maize, rice grains and coffee husk; all of them were inoculated with disks of culture, concentrated liquid media or diluted liquid media (1:40) colonized by P. chlamydosporia. Besides, the substrates milled maize, supplemented or not with potato broth, and inoculated with fungus disks or aqueous suspension were evaluated. Milled maize and coffee husk were the best substrates for chlamydospores production. The best inoculum kinds were disks of culture and diluted liquid media (1:40) colonized by P. chlamydosporia. The supplementation of milled maize with potato broth did not improve the production of chlamydospores, and the best inoculum form was disks of fungus. Moreover, it was also studied the substrates rice grains, coffee husk and coconut fibers with different rates of water; the substrate coffee husk moistened by different periods; the supplementation of coffee husk with rice flour or dextrose; and the substrate grains of rice sterilized in microwave oven with different rates of water. The substrate grains of rice, in all of the amounts of water tested showed increase in production of chlamydospores. There was no difference in the chlamydospores production of the coffee husk substrate, when moistened by different periods of time and only when it was supplemented with rice flour it showed higher mean of clamydospores per gram of substrate. All treatments evaluated showed higher production of chlamydospores in the substrate grains of rice treated in the microwave oven. The best substrate for the production of chlamydospores of P. chlamydosporia was the rice grains

Biocontrol of Pythium in the pea rhizosphere by antifungal metabolite producing and non-producing Pseudomonas strains

The definitive version is available at www.blackwell-synergy.com. Copyright Blackwell Publishing DOI : 10.1046/j.1365-2672.2001.01260.xFour well-described strains of Pseudomonas fluorescens were assessed for their effect upon pea growth and their antagonistic activity against large Pythium ultimum inocula. The effect of Pseudomonas strains upon the indigenous soil microflora, soil enzyme activities and plant growth in the presence and absence of Pythium is assessed. Pythium inoculation reduced the shoot and root weights, root length, and the number of lateral roots. The effect of Pythium was reduced by the Pseudomonas strains as follows: F113, SBW25 and CHAO increased the shoot weights (by 20%, 22% and 35% respectively); strains Q2-87, SBW25 and CHAO increased root weights (14%, 14% and 52%); Strains SBW25 and CHAO increased the root lengths (19% and 69%), and increased the number of lateral roots (14% and 29%). All the Pseudomonas strains reduced the number of lesions and the root and soil Pythium populations, whilst SBW25 and CHAO increased the number of lateral roots. Pythium inoculation increased root and soil microbial populations but the magnitude of this effect was Pseudomonas strain specific. Pythium increased the activity of C, N and P cycle enzymes, whilst the Pseudomonas strains reduced this effect, indicating reduced plant damage. Overall, strains SBW25 and CHAO had the greatest beneficial characteristics as these strains produced the greatest reductions in the side effects of Pythium infection (microbial populations and enzyme activities) and resulted in significantly improved plant growth. Surprisingly strain SBW25 does not produce antifungal metabolites, and its biocontrol activity was related to a greater colonisation ability in the rhizosphere.Peer reviewe

Long-term suppression of Pythium ultimum in arid soil using fresh and composted municipal wastes

7 pages, 4 tables, 4 figures.The effect of addition of municipal solid waste (MSW) at different degrees of stabilisation on the biological properties of an arid soil was studied 24 months after application. This included effects on the indigenous soil microflora and soil enzyme activities in the presence and absence of Pythium ultimum. The addition of organic waste (fresh or composted) reduced populations of culturable bacteria and fungi and disease symptoms caused by P. ultimum, and resulted in heavier plants with longer and more extensively branched roots. Addition of organic waste increased the population size of culturable bacteria and fungi, while enzymatic activity of the soil was higher in soil amended with organic matter than in non-amended soil. Populations of biological control agents, such as Trichoderma and fluorescent pseudomonads, were larger in soil amended with organic matter. The addition of urban waste could therefore be a suitable technique with which to restore soil quality by stimulating biological control against plant pathogens such as P. ultimum.Peer reviewe