Biological Control of Plant Parasitic Nematodes
and Development of Bio-Nematicides
Wang Zhiwei • Weng Zhonghe
(Nanjing Agricultural University • Hainan Holon Technology Co.Ltd.)
1. Plant parasitic nematode problems
Plant parasitic nematodes are parasitic on most part of crops and a plenty of wild
plants. Concurrences of direct injury caused by plant parasitic nematodes and secondary
parasitism of various plant pathogens are recorded on the host plants all over the world.
Especially, in various kinds of greenhouses, plant parasitic nematodes have a longer
lifetime, higher population, and can cause severe damage to horticultural plants. It is
also recognized as a cause of soil-sickness. Plant parasitic nematodes cause big losses in
agricultural production and control of plant parasitic nematodes had become a important
matter in plant protection the world.
In China, most important group of plant parasitic nematodes should be root-knot
nematodes Meloidogyne spp.. Among more than seventy species included in genus
Meloidogyne in the world, the southern root-knot nematode M. incognita (Mi), M.
javanica (MJ), the peanut root-knot nematode M. arenaria (Ma) are most popular
rot-knot nematodes in China. But Mi should be the most widespread one. Meloidogyne
spp. exist in soils from temperate to sub-tropical region, and it invades into root of more
than 700 plant species, including major crops in Cucurbitaceae, Rosaceae, Grammeae
and many others. Meloidogyne spp. Caused severe yield losses on vegetables and fruits
every year. Effective nematicides used up to now such as Furadan and some others are
going to be prohibited because of the pollution of the environment. In fact, Hainan
province has prohibited use of Furadan on vegetable production. On the other hand,
effects of newly provided chemicals are usually not satisfied on their activities of
protection. Now, a lot of farmers are short of effective chemicals for nematode control.
Further more, continuous and large amount application of those harmful chemicals,
especially within greenhouses, makes various troubles, especially in food safety,
pollution of environment, decrease of bio-diversity in the field. A new, effective and
easy-use nematicide is desired. This trend is also recognized all over the world in these
years.
2. Approaches for nematode control
Chemical control, physiological control, resistant plant breeding, crop rotation, and
microbiological control were recommended for plant protection against plant parasitic
nematodes. In modern agricultural systems, especially in horticulture, nematode control
is now mainly depending upon application chemical nematicides in the soil. Abundant
application of nematicides now makes very severe troubles in agricultural production,
food safety and environmental protection. So, a new, effective, environmental friendly
and easy-use novel nematicide is expected. Such kind of nematicide expected to be
developed through biological control research.
Trials of biological control of plant parasitic nematodes are practiced all over the
world. A large number of natural enemies, including parasitic and trapping fungi,
mycorrhizal fungi, bacteria, viruses, nematodes, insects, mites, invertebrates, are
investigated as possible biological control agents for nematode protection. The
existence of nematophagous fungi had influence on nematodes was first noted by Zopf
in 1888. The number of published papers on these organisms probably exceeds the
number of those published on all other biological control organisms combined.
Presently, over 400 species of nematophagous fungi affect on various nematodes in
agricultural soils. In China, Professor Zhang Keqin in Yunnan University published an
excellent specialist book on nematophagous fungi ("Studies on Nematophagoius Fungi",
Yunnan University Press. 2000), and reviewed almost all nematophagous fungi in
China. Nematophagous fungi are mainly divided into two classes: nematode-trapping
fungi and egg-parasitic fungi. During the past years research on the use of biological
agents for controlling nematodes has focused on the application of nematode-trapping
fungi. Recently, egg-parasitic fungi, e.g. Paecilomyces lilacinus have become a new,
attractive biological control agent for plant parasitic nematode control. By using
nematode-trapping fungi, bio-nematicides Royal 300 and Royal 350 were developed for
control of Meloidogyne on vegetables in France. In Philippines, on the other hand,
egg-parasitic fungus Paecilomyces lilacinus was used to development of another
bio-nematicide. Commercial productions of these fungi have used to be practiced in
those countries. Unfortunately, inconclusive results had obtained by other investigators
when those bio-nematicides were used for control of Meloidogyne. In China,
development of bio-nematicides by using nematophagous fungi is actively promoted by
many scientists in universities, institutes, and some companies. The interaction between
nematophagous fungi and nematodes is complex, and activity of these fungi may be
influenced by many factors.
Bacterial agents such as Pseudomonas spp., Bacillus spp., Pasteuria penetrans and
others are also studied for nematode biological control. By using Pasteuria penetrans,
scientists developed a novel bio-nematicide, Pasteuria-Wettable Powder, and
successfully suppressed Meloidogyne incognita at extremely low level.
Pasteuria-Wettable Powder has made a great significance for nematode control in
Japan.
As a candidate of biorational pesticide fro plant parasitic nematode control, some
Japanese scientists pay a great attention to physiological active compounds produced by
Streptomyces sp.. AM (Arbuscular Mycorrhiza) and endophytic fungus are also known
to affect to various plant parasitic nematodes. However, until now, Pasteuria-Wettable
Powder maybe can be calculated as the most successful example of bio-nematiside.
3. Pasteuria penetrans: an obligate parasite of nematodes.
From the first finding of Pateuria sp. from Pratilenchus sp. in 1940, this type of
bacterial has been found from 96 genus 205 species of nematodes and P. penetrans (Pp)
from root-knot nematodes, P. thornei from root lesion nematodes and P. nishizawae
from cyst nematodes are identified. Pp exists in natural water or soil as a kind of
endospore. The spore can adhere to the cuticle surface of secondary-stage juveniles of
Meloidogyne spp.. When the spore germinated, the germ tube penetrates the cuticle
layer, and develops vegetative mycelium within the juvenile. The vegetative mycelium
develops into microcolony, the microcolony the daughter colonies. The daughter colony
then will be fragmented in eventually quartets, doublets and Finally sporangium in
which a single spore formed. Instead of nematode eggs, forth-stage juveniles infected
by pp will be full with about two million Pp spores. This makes rapid decline of
nematode population in the soil, mediating high protecting effect on host plants.
A strict host-parasite relationship has been demonstrated between pp spores and
root-knot nematodes, making this spore difficult to be cultivated on the medium but
extremely safe in the environment. Even many scientists are interested with its potential
as a biological control agent. However, difficulties on large-scale preparation of the
spore prevent them from advanced research and development. This delayed pp
application on plant protection.
4. Development of Pasturia-wettalbe powder .... Our approach .....
Using Pp, the most studied nematode parasitic bacteria, we successfully developed
a bio-nematicide, and now, registration of this new bio-nematicide is going on in
Japanese ministry of agriculture, Forestry and Fisheries. Here, we describe outline of
the development processes:
(1) Large scale preparation
Based on biological characteristics of the Pp, we firstly challenged the
large-scale preparation of Pp spores .we successfully overcame the difficulties
on Pp propagation by mixing pp spores and nematode juveniles, inoculating to
tomato seedlings, and then harvest in tomato roots when the daughter spores
matured.
(2) Excellent effects in both greenhouses and fields
Using spores by propagated on tomato plants, we tested the protection effect of
p spores from nematode injury on tomato, cucumber, pumpkin, sweet potato,
container fig and many other crops in both greenhouses and fields .as shown in
the table, although control effects on 1st and 2nd cropping are still not satisfied,
Pp possess a long, stable and excellent protecting effect on tomato plants from
3rd cropping to 5th cropping. This indicated that pp spores could be applied as
a biological control agent active against to mi when p spores are applied with
chemical nematicide.
(3) Formulation of the pasteuria-wettable powder
Through various tests in greenhouses and fields, it was clarified that spore
dispersal within the soul greatly affects on the protecting activity. According to
this observation, we tested various formulations and finally selected a wettable
powder formulation for best dispersal and named as pasteuria-wettable
powder.
(4) Toxicity and safety
Pasteuria-wettable powder passed toxicological tests on microorganisms,
free-living nematodes, insects, plants, fish and mammals; results of those tests
indicated that Pasteuria Wettable Powder is an extremely safe bio-nematicide
(5) Stability and preservation
Pp spores are stable for 2 or more years at 4°C as suspension, or for 2 or more
years at room temperature as a dry wettable powder, therefore, Pp spores could
be preserved at 4°C in water before formulation and the pasteuria-wettable
powder could be preserved at room temperature. This makes the
pasteuria-wettable powder easy to handling.
(6) Resistance to chemical and environmental stresses
Pp sores are highly resistant to a number of nematicides used in the fields,
making Pp to be possibly used together with chemical nematicides. On the
other hand, once the spores were applied, they can exist, propagate and
maintain their infectivity to nematodes for at least 6 hours in the field. This
causes Pp spores are effective to mi for a long term in the Mi infested field.
(7) Application techniques
Based on the biological and plant pathological properties of Pp spores, an
application draft of pasteuria-wettable powder was recommend: apply 2X10"
spores /m2 with half volume of chemical nematicides in to field, followed by
application of abundant water. Seven to ten days later, the field is in ready to
transplanting.
5. Conclusion
In the natural environment, plant pathogens live and survive in very complex
situation. In conventional agriculture systems, pathogens or insects cause injury at
various conditions, but the severity is usually restricted. These kinds of restriction are
usually due to resistant genes in hosts, natural enemies, crop rotation, mixed cropping
and so on. However, in the modern agriculture systems, these kinds of restriction
usually are broken by crop improvement, abundant application of fertilizers and
pesticides, extension of modern horticulture etc. Pasteuria spp. is considered to be
active against to nematodes from ancient time, and their biological roles are postulated
before scores of years. However, application of this bacterium for agricultural studies is
not available. Our techniques on large-scale propagation and unified dispersal of Pp
spores in soil led us obtained an excellent protecting activity against Mi in the field.
This indicated that each or combination of conventional or basic techniques and
knowledge are very important in biological control.
Effect ofPasteuriapenetrans on plant vigor and fruit harvest of tomato in Japan
Treament
Pasteuria
Chemicals
Control
plant
1st crop.
185
196
125
height (cm)
2nd crop. 3rd crop.
129
(141)
155
168
149
150
fruit weight Cg)
4th crop
5th crop
1405
250
225
3080
1760
1210
Pasteuria: treated with 5X109 spores/m2 of oxamy11 at 1st cropping, and additional
treatment of l0g/m2 of fosthiazate at 5th croppingChemicals: Treated with 30g/m2
ofoxamyl at 1st and 4th cropping.20g/m2 ffosthiazate at 5th cropping
(These results obtained from Nematech. Co. Ltd., Japan.)