FUNGAL BIOTECHNOLOGY

FUNGI IN AGRICULTURAL BIOTECHNOLOGY

An area of agricultural biotechnology in which fungi show considerable potential for the future is the biological control of pathogenic fungi, insects & weeds. Such as biological approach to pest control is attractive because it provides an alternative to chemical pesticides which are increasingly perceived as being harmful to the environment. The second area of agricultural biotechnology where fungi are likely to be important is as inoculates or biofertilizers for use in increasing crop production.

CONTROL OF FUNGAL PLANT PATHOGENS BY MYCOFUNGICIDES -

The species of the genus Trichoderma have become the most widely investigated of all potential mycofungicides. Modern approaches to the use of this fungus to control pathogenic fungi have largely been based on the direct use of inoculants.

Spares of Trichoderma species can be readily & economically produced in the laboratory. But, the fact that no spectacular success has been achieved following the use of Trichoderma as an agent to control fungal pathogen, suggests that either members of this genus are not particularly efficient under field conditions, or more probably that we are not using them to best advantage. Safe & effective biocontrol results have been achieved with Trichoderma under very specialized conditions.

BIOLOGICAL CONTROL -

The use of chemical pesticides, has led to dramatic improvements in the production of crop plants. Since such improvements provided a reliable supply of cheap food they were initially welcomed of late, consumers are becoming increasingly concerned both about food quality & of the real & imagined effects of modern farming methods on the natural environment. While many of these fears have been exaggerated, there is neverthless a developing concensus that modern petro-chemical based farming is ultimately non-sustainable, as a result, more ecological approaches to food production are now being researched.

Pesticides were originally based on toxic heavy metals such as arsenic, mercury, lead or copper. Modern pesticides, however, are organic compounds, with a high degree of specificity towards their target organism. They also generally exhibit low over all toxicity & have little immediate impact on the environment. It is possible that the long term effects of these compounds might be subtly detrimental to soil fertility. Despite the positive results of the use of modern pesticides, concern continues to be expressed about the wisdom of using large quantities of chemicals in the environment. These fears have led biotechnologists to examine alternatives to chemical pesticides as a means of controlling agricultural pests. The most obvious, and apparently environment- friendly, alternative to pesticides is to use naturally occuring biological approaches to the control of pest populations.

Since all pests have natural antagonists, biological control should in theory be relatively straightforward. In practice, successful biological control is extremely difficult to achieve.

Fungi possess a number of characteristics that make them potentially ideal biocontrol agents. Firstly, many saprophytic species antagonise, representatives of all the pest organisms, including plant pathogenic fungi, weeds & insects secondly, fungi can be readily grown in culture so that large quantities can be economically produced for release, mainly as spores or mycelial fragments, into the environment. These inoculants then germinate or grow to produce active mycelium which can parasitise or otherwise inhibit the pest without damaging the non-target organisms. Fungi also survive for relatively long periods as resting bodies, and can then germinate to grow & control the target population thereby making continual reinoculation with the biocontrol agent unnecessary.

Table - Examples of biocontrol agents used commercially or in near commercial condition against soil borne or root-infecting pathogens. Control agent Disease Crop 1. Trichoderma harzianum White rot Onion 2. Phlebia gigantea Heterbasidion root rot Pine 3. Agrobacterium radiobactor var. radiobactor Crown gall Rose 4. Sporidesmium sclerotivorum Lettuce drop Lettuce 5. Talaromyces flavus Damping off Sugarbeet

USE OF FUNGI TO CONTROL INSET PEST -

Over 400 species of fungi attack insects & mites, so there is great potential for the use of these organisms as biological insecticides. As insect biocontrol agents, fungi are markedly superior to other micro-organisms because they are generally non-specific in their action & are useful against a wide range of insect pests. On the debit side, however, their use is limited by the restricted range of humidities in which they can grow,. An absolute humidity in which they can grow, an absolute humidity greater than 90% for a relatively long period is needed to enable fungi to grow & infect insect hosts. Another disadvantage is that fungi rarely, if ever, kill insects rapidly in the manner that we have come to associate with chemical insecticides.

Most of the so-called entomopathogenic fungi are phycomycetes & Deuteromycetes. Spores of these fungi attack either the external or gut cuticle of their insect hosts. They then germinate & hyphae penetrate the haemocoel. Death may result from the production by the fungus of a toxin, or following the direct utilization of the body fluids. After the insect body reserves have been used up, the mycelium then dies from lave of substrate & sporulates, the spare mass then acts as a focus of future infection.

Insecticidal toxins produced by fungi are non-enzymic, low molecular weight products, which can kill insects when present even at low concentrations. The best examples of the use of fungi to control insects are provided by species of Beauveria & Metarhizium, fungi which respectively cause white & green muscardine disease of insects.

The ideal mycoinsecticide should rapidly penetrate the cuticle of the insect, produce toxins to induce rapid paralysis, rapidly colonise the insects body & then sporulate under most environmental conditions. An obvious problem is that fungi used to control insects may be killed by fungicides sprayed on insect- infested crops.

Table - Principal Deuteromycete fungal candidates for arthropad.

	SPECIES	MAIN TARGET PESTS
1.	Aschersonia aleyrodis	White fly
2.	Beauveria bassiana	Colorado beetle
3.	Beauveria brongniartii	Cockchafer
4.	Hirsutella thompsonii	Rust mites
5.	Metarhizum anisopliae	Beetles,bugs
6.	Nomuraea rileyi	Caterpillars
7.	Verticillium lecanii	Aphids, Whitefly

COMMERCIAL PRODUCTION OF FUNGAL INSECTICIES -

Fungi can be readily produced for use as fungicides, as they are easy to grow on a variety of substrates, including agar-based media, cereal grains & a variety processed & unprocessed wastes; cereal grains are generally considered the best & most economical substrates for this purpose.

Product self-life is a major limitation of fungal biocontrol agents. A period of 18 months survival at 20° C is thought to be optimal, although production methods in current use can only achieve survival rates of around 3-6 months at storage temperatures of 40C Biocontrol fungi are usually mixed or formulated with substrates to increase their survival, in some cases colours or chemicals may be added to attract the insect host to the fungal pathogen.

USE OF FUNGI TO CONTROL NEMATODES

Fungi that parasitise nematodes (nematophagus fungi) can be divided into nematode trapping fungi, endoparasitic species & fungi that parasitise nematode eggs. Nematode trapping fungi capture nematodes with specialised structures such as constructive & non-constructive rings, adhesive knobs or lastly by producing an adhesive material along the entire mycelial surface. Endoparasitic nematophagous fungi line in soils where they produce adhesive spores. These become attached to body of the nematode, on germination, a germ tube enters the body where it grows & consumes the host. Egg parasites, as their name suggests, are nematophagous fungi that parasitise the eggs of nematodes.

The following approaches have been used in an attempt of use fungi to control nematodes: 1) Exploitation of naturally supressive soils.
2) Soil amendments to encourage indigenous bio-control agents.
3) Inoculation of soils with selected strains of bacterial & fungi.
4) The use of microbal toxins & enzymes

1) Supressive soils-

Certain soils are naturally supressive to nemotodes. Fungi implicated in naturally supressive soils include Nematopthasa gynophila, Verticillium Chlamydosporium & Dactylella Oviparasitica.

2) Soil amendments -

A wide range of amendments have been found to be effective in controlling soil nematode populations. These generally involve breakdown products of added organic materials, including fatty acids & ammonia which together with an increase in natural antagonists prove effective in reducing nematode populations. Chitin amendment has been particularly extensively studied as a means of increasing populations of specific nematods antagonists.

3) Application of selected fungi -

To date, Paecilomyces lilacinus is the only fungus that has been widely tested for nematode control under field conditions.

4) Enzymes & toxins -

The direct application for enzymes, such as chitinase from egg parasitising fungi & collagenase from nematode - trapping species, to soils may also be useful means of controlling nematodes. Potentially useful nematocidal toxins are produced by fungi such as the oyster mushroom, pleurotus ostreatus.

USE OF FUNGI TO CONTROL WEEDS:

Weeds, in the widest sense of the world, include algae, ferns, annual & perennial plants, and also herbaceous or woody species. Fungi that are potential mycoherbicides can be divided into facultative parasites, which can be grown in the laboratory, and obligate parasites which at present cannot.

Two approaches to used control using fungi have been developed (i) the inundative or mycoherbicide approach, and (ii) the classical biocontrol method. In the former approach, massive inoculations of fungi are applied at the optimum time for infection. The biocontrol agent then persists & controls the weed population.

The classical method of biocontrol is achieved over a longer time period & depends upon the gradual increase in disease, the long term development of the biocontrol agent means that significant weed control may take months or even years.

USE OF FUNGI TO PRODUCE CHEMICAL PESTICIDES & BIO-FERTILIZERS -

Biologically active secondary metabolitises produced by fungi are being evaluated as potential pesticides, particularly for use in controlling plant growth. These compounds have the advantage over convential pesticides in being effective at very low concentrations while proving essentially non-persistant & harmless to the environment.

USE OF FUNGAL INOCULANTS TO IMPROVE CROP GROWTH -

Many of soil bacteria & fungi can liberate plant- available phosphorus from insoluble phosphates. This ability is usually associated with the production of organic acids & chelating agents, including citric acid & 2 - cetogluconic acid . Insoluble phosphates are found in abundance throughout the world, mainly as rock phosphate. Since rapidly available phosphate is desirable, the release of phosphorous, from these materials by natural weathering is far too slow to be a general benefit in agriculture. The rate of such phosphate release can, however, be stimulated by inoculating a microbial phosphate solubiliser into the soil. This could be a heterophilic bacterium, a member of the genus, Thiobacillus, or a phosphate solubilising fungus. Since fungi are heterotrophs, a nutrient source has to be added together with the inoculant & insoluble phosphate. Suitable carbon sources include molasses & sugarbeet bagasse.

MYCORRHIZAL FUNGI AS INOCULANTS FOR USE IN IMPROVING CROP GROWTH -

Mycorrhizas are symbiotic associations between soil fungi & most higher plants. It was soon recongnized that mycorrhizal infection can often greatly increase the rate of uptake of nutrients such as nitrogen & phosphorus from nutrient-deficient soils. This has led to the view that the inoculation of mycorrhizal fungi into soils should lead to an increase in the uptake of these essential plant nutrients.

Two types of mycorrhiza have been recongnized, the endotropic or vesicular -arbuscular mycorrhiza (VAM) , & the ectotrophic type. In VAM the fungal partner is restricted to the cells of the plant cortex where it grows within & without the cells, invaginating the host cells at intervals to form a dichotomously branched structure called the arbuscle, thought to be the site of nutrient exchange between plant & fungus. The fungal partner appears to have no independent existence is soil. Neither is the interactioin specific, since a single species of fungus can infect a wide range of plant, including most crop species. It is for this reason that VAMS are economically the most important type of mycorrhizal association.

In ectoirophic mycorrhizas, the fungal partner forms a tight sheeth around the plant root & from this sheath hyphae grow into the outer cortex to form a network called the Hartig net. Ectotrophic mycorrhizas, unlike VAMS , tend to be non-specific. The fungal partner is readily grown in culture, and forms symbioses with trees in temperate forests.

Vesicular-arbuscular mycorrhizas can directly enhance the uptake by plants of essential nutrients such as phosphorus, copper & iron on the other hand, Zinc & manganese uptake may be reduced, so that the mycorrhizal association many protect some plants from the toxic effects of the larger amounts of these elements. Ectotrophic mycorrhizas also show enhanced uptake of phosphorus, and by mineralising organic nitrogen make nitrogen available for plant use. They may also protect their plant hosts from heavy metals & attack by pathogens, and they may also help increase the uptake of water from soil to plants.

SOME BIOTECHNOLOGICALLY IMPORTANT FUNGI

FILAMENTOUS SPECIES -

Most of the filamentous fungi that are important in biotechnology are members of the important in biotechnology are members of the Deuteromycetes (Deuteromycotina). Examples include Aspergillus niger, pencillum notatum - chrysogenum & Trichoderma Viridae. Basidiomycetes white rot fungi such as phanerochaete Chrysosporum are becoming increasingly important in environmental biotechnology because they are able to metabolise a variety of organic chemicals, many of which are pollutants.

-ASPERGILLUS SPP. -

There are about 200 species of Aspergillus, found throughout the world growing on a vast array of substrates. Aspergillus niger, as its specific name suggests, is a black fungi which is commonly called 'black mould'. Many Aspergilli contaminate foods, producing toxic products;A flavours for example, produce the mycotoxin alfatoxin. Several species also grow as contaminants on leather & cloth. Aspergillus species are important in medicine, causing disease of the internal organs. Aspergillus fumigatus accounts for nearly all of these infections. Aspergillus species produce numerous extracellular enzymes, many of which are put to good use in biotechnology. Aspergillus niger is particularly important in the manufacture of citric acid & gluconic acid, both finding wide application in food industry.

The asexual stage of Aspergillus is the one most often encountered by biotechnologists.

-PE NICILLIUM SPP. -

Penicillin species are as widespread & cosmopolitan as the aspergilli. They are frequently referred to as green or blue moulds & are often found contaminating citrus fruits or causing decay on refrigerated cheese & other foodstuffs. Various species are also pathogenic to citrus fruits. Like members of the genus Aspergillus, penicillin species also attack leather & fabrics. The production of penicillin is without doubt the most significant use of this fungus. The only other therapeutically useful antibiotic produced by members of this genus is griseofulvin, which is used to treat fungal infections of the skin, including athelet's foot. Penicillium species also produce organic acids & flavours cheeses.

- TRICHODERMA SPP. - Trichoderma species are ubiquitous soil fungi which produce white, yellow or green colonies when cultured. Trichoderma species are used to produce cellulases. They are particularly effective as antagonists of the growth of other fungi, many of them plant pathogens, with the result that trichoderma species are important biocontrol agents.

- PHANEROCHAETE CHRYSOSPORIUM - It is a member of the Basidiomycetes but it is the filamentous stage rather than the sporophore that is used in biotechnology. It belongs to a group called the white rot fungi, a name that emphasises the importance of these fungi as agents of wood decay. It is noteworthy for its ability to produce non-specific ligninases which can be used to degrade pollutants both in liquid effluents & in soils.

- YEASTS -

Yeasts play a fundamentally important role in biotechnology. The classic yeasts are unicellular & are slimy in appearance when growing on media often resembling bacterial colonies. Saccharomyces species are widely used in the food industry in bread production & in the fermentation of alcoholic beverages. Yeasts have also been used as source of single-cell protein, and to produce alcohol for industrial purposes. Recent developments in the molecular biology of yeasts have significantly expanded the biotechnological potential of these fungi.

-TABLE- INDUSTRIAL APPLICATIONS OF YEASTS ORGANISM USE 1. Saccharomyces Cerevisiae Brewing, baking, fuel, alcohol,wine 2. Saccharomyces uvarum Brewing, melibiase 3. Ashbya gossypii,Eremothecium ashbyii Riboflavin 4. Rhadotorula Carotene 5. Hansenula, Pichia SCP Via methanol 6. Yarrowia Lypolytica Citric acid 7. Saccharomycopsis,Aureobasidium D-Gluconic acid

- ASARICUS SPP. - Agarius spp. are almost exclusively used in biotechnology for the production of mushrooms i.e. as a protein rich foodstuff. The commercial species used in mushroom growing in almost exclusively A bisporus.

- CLAVICEPS PURPUREA -