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 isolate

The 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. chlamydosporium

The 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 general
smaller 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 study

The 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.