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BIOLOGICAL CONTROL OF PLANT PATHOGENS
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Root
Diseases (mycoparasites) |
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Root
Diseases (antagonists) |
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Root
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Introduction The biological
control of plant pathogens was detailed by Van Driesche & Bellows (1996).
It involves the ecological management of a community of organisms. In the
case of plant pathogens, however, there are two distinctions from biological
control of organisms such as insects and plants. First, the ecological
management occurs at the microbial level, typically in microcosms of the
ecosystem such as leaf and root surfaces (Andrews 1992). Second, biological
control agents include competitors, as well as parasites. While
hyperparasites of plant pathogens and natural enemies of nematodes function
in much the same way as do parasitoids, in arthropod systems (by destroying
the pest organisms), competitors function by occupying and using resources in
a nonpathogenic manner and in so doing exclude pathogenic organisms from
colonizing plant tissues. Microbes which negatively affect pathogenic
organisms are called antagonists. Diseases of
roots, stems, aerial plant surfaces, flowers, and fruit are caused by a wide
variety of pathogens. Because of this diversity, the antagonist species which
negatively affect plant pathogens and the mechanisms by which they accomplish
their beneficial action are also quite varied. Their biological and taxonomic
diversity is covered in some detail in several texts and reviews, including
Cook and Baker (1983), Fokkema and van den Heuvel (1986), Campbell (1989),
Adams (1990), and Stirling (1991). This section briefly introduces the
antagonists of some important plant pathogens as representative of the broad
taxa which are important in this field, beginning with agents affecting
microbial pathogens of roots, and proceeding through pathogens of stems,
leaves, flowers, and fruit. Natural enemies of plant parasitic nematodes are
treated in the last section. Root Pathogens Root diseases
are caused by a wide variety of fungi, and by some bacteria, in many crops
and plant systems. Biological control agents recognized as significant in
suppression of these diseases are largely antagonists which can occupy niches
similar to the pathogens and either naturally or through manipulation out
compete the pathogens in these niches. Antibiotic production is also
important in a few cases, as are mycoparasitism and induced resistance. Streptomyces
scabies, the causative organism of potato scab, is suppressed by naturally
occurring populations of Bacillus subtilis, and saprotrophic Streptomyces
sp.). Other microorganisms recognized as suppressing fungal diseases
include species of Pseudomonas and Bacillus. Saprotrophic
Fusarium fungi are able to suppress populations of pathogenic Fusarium
spp. through competition for nutrients. There are few well-documented cases
of induced resistance for soil-borne pathogens, and these are mostly of wilt
diseases. Examples of organisms that induce resistance in plants to pathogens
include nonpathogenic strains of Fusarium spp. Verticillium spp. &
Gaeumannomyces spp. Mycoparasitic flora such as Anthrobotrys pp,
Coniothyrium minitans Campbell and Sporidesmium scerotivorum
Uecker et al., can be added to soil against fungal diseases. Bacillus spp.
and especially Pseudomonas spp. are among bacteria that have
properties particularly suited to effective suppression of root-infecting
pathogens in soil, such as antibiotic production and competition for Fe3+
ions. Mycetophagous soil amoebae have also been noted feeding on pathogenic
fungi. These amoebae generally require moist conditions in which to function,
and may be important in the natural control of some fungi. Stem Pathogens Diseases of
plant stems produce symptoms which include decay and cankers on forest and
orchard trees. and such wilts @ts I)utch elni disease and chestnut blight
(caused by the fungus Cryphonectria parasitica (Murrill) Barr of Asian
origin infecting the American chestnut. Castanea dentata [Marshaml Borkjauser).
Because the etiologies of stem diseases vary, the taxa involved in biological
control also vary. In many stem diseases, the pathogen colonizes a part of
the host which initially is relatively free of microorganisms, such as a
pruning wound. Successful biological control in such circumstances depends on
rapidly colonizing this pristine environment with a nonpathogenic
antagonistic competitor (VanDriesch & Bellows 1996). Primary among these
are competitively antagonistic fungi, including saprotrophic members of the
genera Fusarium, Cladosporium, Trichoderma, and Phanerochaete, and
such antibiotic producing bacteria as Bacillus subtilis and Agrobacterium spp.
In the case of chestnut blight, hypovirulent strains of the pathogen itself
are crucial in bringing about biological control. In this case, hypoviruience
is transmitted cytoplasmically to virulent strains already infecting trees,
and disease symptoms decline and disappear (VanDriesch & Bellows 1996). Leaf Pathogens The growth of
microorganisms on leaves is normally severely restricted by environmental
factors. Nutrient levels generally are low on leaf surfaces, and microclimate
variables, especially leaf surface moisture, temperature, and irradiation,
are often unfavorable for microbial development. In temperate climates and
arid tropical regions, water will be intermittent on leaf surfaces, but may
be continually present in humid tropical regions. Temperatures on leaf
surfaces exposed to direct radiation may rise to several degrees above
ambient, The result of such variation is that microbial floral development on
leaf surfaces varies from general scarcity in temperate climates to more
extensive microbial films in tropical rain forests (Campbell 1989). Microbes that
most frequently are recorded as saprotrophs on surfaces of crop plants in
temperate conditions and, therefore, the species which are candidates as
antagonists of pathogens, include the fungi Aureobasid m pullulans (de
Bary) Arnaud, Cladosporium spp., and such yeasts as Cr.yptococcus spp.
and Sporobolomyces spp. Beneficial bacteria in the phyllosphere
include members of such genera as Erwinia, Pseudomonas, Xanthomonas,
Chromobacterium and Klebsiella. These lists,based on microbial surveys,
usually give no indication of activity of the organisms, but this information
can be obtained from experimental studies. For example, early studies on
control of botrytis rot in lettuce (Wood 1951) indicated that several
organisms were successful in suppressing the disease when sprayed on lettuce (Lactuca
sativa L.) plants, among them Pseudomonas sp., Streptomyces sp.,
Trichoderma viride Persoon: Fries, and Fusarium sp. Similar
studies show varying degrees of effectiveness in other cropping systems (Peng
& Sutton 1991; Sutton & Peng 1993a,b; Zhang et al. 1994). The
microbial composition and biological activity of phylloplane microbes can
vary with season, position on the top or bottom of the leaf and on location
in the plant canopy, depending on the degree of exposure relative to prevailing
winds and rain (Campbell 1989). Biological
control of the black-crust pathogen (PhyIlacbora huberi Hennings) on
rubber tree (Hevea brasiliensis Müller Argoviensis) foliage is
accomplished by the hyperparasites Cylindrosporium concentricum Greville
and Dicyma pulvinata (Berkeley & Curtis) Arx (Junqueira &
Gasparotto l991). Botrytis leaf spot in onion (Allium cepa L.) was
suppressed by Gliocladium roseum Link: Bainier (Sutton and Peng
1993a). Other examples include control of powdery mildews, other botrytis
rots, and turfgrass diseases (Sutton and Peng 1993a). Nonpathogenic
species of the fungal genus Colletot richum 1981; Dean & Kuc 1986)
can be used to induce resistance in cucumbers against pathogenic species of
the same genus. Inoculation with a nonpathogenic strain of a virus confers
protection to plants from pathogenic strains in many diseases. The bacterium Bdellovibrio
bacteriovorus Stolp & Starr is aparasite of pathogenic bacteria.
Finally, there are numerous parasitic fungi which attack pathogenic fungi
(Kranz 1981). Among those which have been studied in detail, principally as
agents against leaf rusts and mildews, are Spbaerellopsis filum
(Bivona-Bernardi ex Fries) Sutton, Verticillium lecanii (Zimmerman)
Viegas, and Ampelomyces quisqualis Cesati ex Schlechtendal (VanDriesch
& Bellows 1996). Flower & Fruit Pathogens Flowers are
ephemeral structures and as such have limited opportunity to become infected.
One major disease of flowers which has received attention is fire blight of
rosaceous plants, caused by the bacterium Erwinia amylovora (Burril)
Winslow et al. Biological suppression of the disease has been achieved
through use of the nonpathogenic species Erwinia herbicola (Lohnis)
Dye (Beer et al. 1984; Lindow 1985b), sometimes in combination with Pseudomonas
syringae van Hall. rwinia
herbicola was used successful by spraying aqueous suspensions of it onto
the flowers just before the time of potential infection (Campbell 1989). The
mode of action is primarily competitive exclusion, with the antagonist
competing with the pathogen for a growth limiting resource and possibly other
effects such as induced cessation of nectar secretion or accumulation of a
host toxin (Wilson and Lindow 1993a). The diseases
attacked through biological control include diseases of fruit on the plant
and post-harvest diseases. One of the first systems developed was against Botrytis
cinerea Persoon Fries in vineyards, where sprays with spore suspensions
of the antagonist Ttrichoderma barzianum Rifai were effective in
suppressing disease incidence. Several organisms, including Gliocladium
roseum, Penicillium sp., Trichoderma viride, and Colletotrichum
gloeosporioides were as effective as fungicides in suppressing B.
cinerea on strawberries (Peng and Sutton 1991). A number of other
examples also have been reported (Sutton and Peng 1993a). Post-harvest
diseases, which can be responsible for 10-50% loss of produce (Wilson and
Wisniewski 1989; Jeffries and Jeger 1990), have received considerable
attention. Numerous reports deal with suppression of post-harvest disease in
fruit crops (Campbell 1989; Wilson and Wisniewski 1989; Jeffries and Jeger
1990) by such organisms as species of Penicillium, Bacillus, Trichoderma,
Debaryomyces, and Pseudomonas. The mode of action of many of these is
generally antagonism, often through the production of antibiotics which
reduce the longevity and germination of spores of pathogens. Others appear to
suppress pathogen growth through nutritional competition or induction of host
resistance (Wilson and Wisniewski 1989). Postharvest rots include major
diseases caused by Botrytis cinerea, Rhizopus spp., and other fungi in
several crops. Competitive and parasitic fungi, including Tiichoderma
spp., Cladosporium herbarum (Persoon: Fries) Link and Penicillium
spp., give control as good as commercial fungicides. Enterobacter cloacae (Jordan)
Hormaeche and Eduards reduces rots liy Rhizopus spp., but there are
restrictions in its use on uncooked food products (Van Driesche & Bellows
1996). Plant-parasitic Nematodes Plant-parasitic
nematodes inhabit many soils and attack the roots of plants. They are
affected by a range of natural enemies, including bacteria, nematophagous
fungi, and predacious nematodes and arthropods. There is some limited
evidence for virus association with nematodes (Loewenberg et al. 1959), but
the etiology of these viruses is not well-known (Stirling 1991). The
biologies of natural enemies of nematodes was reviewed by Sayre and Walter
(1991) and Stirling (1991). Bacteria That Affect Plant-Parasitic
Nematodes A few bacterial
diseases of nematodes have been reported (Saxena and Mukerji 1988); other
bacteria produce compounds that are detrimental to plant-parasitic nematodes
(Stirling 1991). The most widely studied of the bacterial pathogens of
nematodes are in the genus Pasteuria. Early work was focused on Pasteuria
penetrans (Thorne) Starr and Sayre. Recent evidence indicates that
this taxon represents an assemblage of numerous pathotypes and morphotypes,
and probably represents several taxa (Starr and Sayre 1988). This bacterium
has been found infecting a large number of nematode species (more than 200 in
about 100 genera, Sayre and Starr 1988; Stirling 1991), does not attack other
soil organisms, and is the most specific obligate parasite of nematodes
known. Its spores attach to and penetrate the nematode cuticle. Most
attention has been centered on populations (Pasteuria penetrans sensu
stncto, Start and Sayre 1988) that attack root-knot nematodes (Meloidogyne
spp.). The spores of P. penetrans germinate a few days after a
contaminated nematode begins feeding on a root (Sayre and Wergin 1977). The
bacterium reproduces throughout the entire female body, and the female may
either be killed or may mature but produce no eggs. Bacterial spores (about 2
million from each infected nematode, Mankau 1975) are released when the
nematode body decomposes, and they remain free in the soil until contacted by
another nematode. They tolerate dry conditions and a wide range of
temperatures, and may remain viable in the soil for more than six months.
Because it is an obligate parasite, it has not yet been possible to develop in
vitro culturing techniques for this bacterium. Different populations of the
bacterium show varying degrees of specificity to small numbers of nematode
species, but the mechanisms and degree of specificity remain to be elucidated
(Stirling 1991). Pasteuria penetrans appears responsible for some
cases of natural regulation of nematode populations (Sayre and Walter 1991). Some strains of Bacillus
tburingiensis are also known to have activity against nematodes,
including plant-parasitic species. Zuckerman et al. (1993) report efficacy of
a strain against Meloidogyne incognita (Kofoid and White) Chitwood, Ratylencbus
reniformis Linford and Oliveira, and Pratylenchus penetrans Cobb
in field and glasshouse trials. The body openings of these nematodes are too
small to permit the ingestion or other ingress of the bacterium, and
Zuckerman et al. (1993) suggest that the mode of action is either a beta
exotoxin (Prasad et al. 1972; Ignoffo and Dropkin 1977) or a delta endotoxin
released following bacterial cell lysis. A strain of B. thuringiensis with
a nematotoxic delta endotoxin is the subject of a European Patent Application
by Mycogen Corporation of San Diego, California (Zucherman et al. 1993). Fungi That Affect Plant-Parasitic
Nematodes Many fungi
attack nematodes in the soil (Barron 1977; Stirling 1991). Numerous species
have been reported from all types of soils. The taxonomy of the group has
been subject to revision, and the generic names recognized in Stirling (1991)
are used here (Van Driesche & Bellows 1996). Some
nematophagous fungi are endoparasitic in nematodes. Among these are genera
which reproduce through motile zoospores (e.g., Catenaria anguillulae
Sorokin, Lagenidium caudatum Barron, Aphanom.yces sp.), which
generally appear only weakly pathogenic in healthy nematodes (Stirling 1991).
Other endoparisitic fungi possess adhesive conidia, and the infection process
begins when conidia adhere to a nematode's cuticle (e.g., the genera Vellicillium,
Drechmeria,Hirsutella, Nematoctonuss). In Nematoctonus spp.,the
germinating spores secrete a nematotoxic compound which causes rapid
immobilization and death of nematodes (Giuma et al. 1973). A few species (Catenaria
auxila [Kuhn] Tribe, Nematophthora gynophila Kerry and Crump) parasitize
adult females or nematode eggs rather than juveniles. Other fungi
capture nematodes through use of special trapping stmctures, and have been
termed "predatory." Among the more common of these fungi are
species in such genera as ,Monacrosporium, Arthrobotrys, and Nematoctonus.
These fungi consist of a sparse mycelium, modified to form organs capable
of capturing nematodes. These organs include adhesive structures, such as
adhesive hyphae, branches, knobs, or nets (Stirling 1991). There are also
nonadhesive rings, the cells of which expand when touched on their inner
surface, constricting the interior of the ring and trapping nematodes. Most
of these fungi are not specific and attack a wide range of nematode species.
They are widely distributed (Gray 1987, 1988) and most are capable of
saprotrophic growth, but often appear limited in this phase in the soil. Many
soils suppress the growth of these fungi (a condition called soil fungistasis
or mycostasis). This is possibly due to two different causes. Mankau (1962)
concluded that a water-diffusible substance was responsible for inhibited
germination in tests of soil from southern California (U.S.A.). Other studies
have indicated increased activity following soil amendments with nutrients
(Olthof and Esrey 1966) or organic material (Cooke 1968), which implies
fungistasis may be a result of resource limitation. Following saprotrophic
growth, formation of trapping structures occurs which is apparently
stimulated by nematodes (Nordbring-Hertz 1973; Janssen & Nordbring Hertz
1980). Stirling (1991) suggests that this phase of predacious activity is
followed by diversion of resources to reproduction, followed by a relatively
dormant phase (Van Driesche & Bellows 1996). Other fungi are
facultatively parasitic on nematodes. Of the few of these fungi that are
significant pathogens of root knot and cyst nematodes, Verticillium spp,
are among the most important. These fungi can parasitize nematode eggs, and Verticillium
chlamydosporium Goddard plays a major role in limiting multiplication of Heterodera
avenae Wollenweber in English cereal fields (Kerry et at. 1982a,b). Paecilomyces
lilacinus (Thom) Samson parasitizes eggs of Meloidogyne incognita (Jatala
et al. 1979) and Heterodera zeae Koshy, Swarup, and Sethi (Dunn 1983;
Godoy et al. 1983). Dactylella oviparasitica Stirling and Mankau, a
parasite of Meloidogyne eggs, is thought to be at least partially
responsible for the natural decline of root-knot nematodes in Californian
peach orchards (Stirling et al. 1979). Predacious Nematodes That Affect
Plant-Parasitic Nematodes Predatory
nematodes are found in four main taxonomic groups: Monochilidae,
Dorylaimidae, Aphelenchidae and Diplogasteridae. Each possesses a distinct
feeding mechanism and food preferences (Stirling 1991). The monochilids have
a large buccal cavity that bears a large dorsal tooth; all species are
precdacious, feeding on protozoa, nematodes, rotifers, and other prey, which
may be swallowed whole, or pierced and the body contents removed. The
dorylaimidss are typically larger than their prey and possess a hollow spear
which is used either to pierce the body of the prey or to inject enzymes into
the food source and suck out the predigested contents. The group is
considered omnivorous. but the feeding habits are known only for a few
species (Ferris and Ferris 1989). Almost all the predatory aphelenchids are
in the genus Seinura. Although small, they can feed on nematodes
larger than themselves by injecting the prey with a rapidly-paralyzing toxin
through their stylet. The diplogasterids, typically a bacteria-feeding group,
have a stoma armed with teeth, and the species with large teeth prey on other
nematodes. Species in all these groups are generally omnivorous, feeding on
free-living as well as plant parasitic nematodes. The role of individual
species in the population dynamics of plant parasitic nematodes in the soil has
been difficult to quantify, but it is possible that a number of species
may act together to produce a significant impact (Stirling 1991). Insects and Mites Several
microanthropods in the soil, including mites and Collembola, prey on
nematodes, and high predation rates have been recorded in vitro (Stirling
1991). A few genera are obligate predators of nematodes, while other genera
are more general feeders and consume nematodes as well as other foods (Moore
et al. 1988; Walter et al. 1988; Sayre and Walter 1991). The
information available suggests that as a group, microanhropods are probably
significant predators on nematodes in some soils and habitats. However,
limited information about predation rates in soil is available, and more work
is required to assess the impact of this group on nematode populations. VanDriesch & Bellows (1996) concluded that this overview
touched briefly on groups of organisms which are antagonistic to plant
pathogens and nematodes. These antagonists vary both in their innate ability
to suppress plant pathogens and in their ability to thrive and compete in
different environments. Consequently the selection of an organism or
organisms for any particular biological control program is a compromise among
these parameters and abilities. In addition, the selection of organisms
depends on the approach taken for their use (inoculative augmentation,
inundative augmentation, or natural control through conservation. Methods For Biocontrol of Plant
Pathogens Organisms for
biological control of plant disease can be used in various ways, but most
attention has been given to their conservation and augmentation in a
particular environment, rather than to the importation and addition of new
species as is often done for insect or weed control. The choice of these
approaches is in part because there is usually a diverse set of microbes
already associated with plants. These microbes provide substantial
opportunity for development of resident species as competitors or antagonists
to pathogenic organisms. Both conservation and augmentation have some
application in each of the main groups of plant diseases. The use of microbes
for control of plant pathogens is covered in more detail in several texts,
including Cook and Baker (1983), Parker et al. (1983), Fokkema and van den
Heuvel (1986), Lynch (1987), Campbell (1989), and Stirling (1991) and in
other review articles (Wilson and Wisniewski 1989; Adams 1990; Jeffries and
jeger 1990; Sayre and Walter 1991; Andrews 1 92; Cook 1993; Sutton and Peng
1993a). Plant pathogens
are attacked with biological control through conservation is accomplished
either by preserving existing microbes which attack or compete with pathogens
or by enhancing conditions for their survival and reproduction at the expense
of pathogenic organisms. Conservation is applicable in situations where
microorganisms important in limiting disease causing organisms already occur,
primarily in the soil and plant residues but in some cases also on leaf
surfaces. They may be conserved by avoiding practices which negatively affect
them (such as soil treatments with fungicides). The soil environment may be
enhanced for some beneficial organisms through adding organic matter, such as
soil amendments (Van Driesche & Bellows 1996). Biological
control of plant pathogens through augmentation is based on mass culturing
antagonistic species and adding them to the cropping system. In the context
of the examples discussed in this text, this is augmentation of natural enemy
populations, because the organisms used are usually present in the system,
but at lower numbers or in locations different than desired. The purpose of
augmentation is to increase the numbers or modify the distribution of the
antagonists in the system. In some cases, such organisms are taken from one
habitat (for example the soil) and augmented in another (for example the
phyllosphere). Tire activity of augmenting microbial agents is sometimes
termed "introduction" in the plant pathology literature, in the
sense of "adding@ them to the
system (Andrews 1992; Cook 1993). However, he organisms introduced are
usually found in a local ecosystem and are not introduced from another region
of the world. Augmentation of
antagonists naturally involves two approaches. The first is direct
augmentation, at potential infection sites or zones, with organisms
antagonistic or parasitic to the pathogens themselves. In this approach, the
antagonist population is directly responsible for disease suppression. A
second approach is to inoculate plants with nonpathogenic organisms that
prompt general plant defenses against infection by pathogens (induced
resistance). Disease control is then achieved through greater plant
resistance to infection. Substantial work
has been done to characterize the role of microorganisms in biological
control of plant diseases. The biological mechanisms underlying the success
of these antagonists in such settings may include initial competition for
occupancy of inoculation sites, competition for limiting nutrients or
minerals, antibiotic production, and parasitism (Van Driesche & Bellows
1996). Characteristics
of the Habitat Understanding
the principles that apply to biological control of plant pathogens, the
ecology of the system is considered at the level of the pathogens and the agents
used for control. Aerial plant surfaces, usually present hostile environments
to colonizing microbes, in many cases consisting of surfaces protected by
cuticular waxes, with very small amounts of nutrients available on these
surfaces. Further, surfaces of the above-ground portions of plants may be
dry. Consequently, pathogenic microbes attempting to colonize these surfaces
may face a number of difficulties, including competition with other,
nonpathogenic, microbes. The rliizosphere
(the roots and the region immediately adjacent) is a somewhat richer
environment than the phyllosphere because of simple sugars, amino acids, and
other materials exuded by the roots, but in the remainder of the soil the
growth of microbes is often carbon limited (Campbell 1989), Moisture in the
rhizosphere may be more continuous in time and space than on the above ground
surfaces of plants (the phylloplane), but the rhizosphere may be subject to
periodic drying. Some forms of
competition in these environments are important to the ability of any
particular organism to increase in numbers and consequently to reduce the
numbers or activity of other organisms, including plant pathogens (Campbell
1989; Andrews 1992). Microbial competition can be important at two main
stages of growth of pathogen populations. First, there may he competition
during initial establishment on a fresh resource that was not previously
colonized by microorganisms. Second, after initial establishment, there is
further competition to secure enough of the limited resources present to
permit survival and eventual reproduction. Microorganisms show many traits
which may characterize them as particularly adept at either the colonization
phase or subsequent phases of competition. Species referred to as r
strategists (ruderal species) have a high reproductive capacity. These
species produce so many spores or reproductive bodies that there is a high
likelihood that some will be found near any newly available resource. These
species are effectively dispersed and establish readily in disturbed habitats
or in the presence of noncolonized resources. They are found in disturbed
settings where easily decomposable organic matter or root exudates are found,
and where initial resource capture is crucial for survival. In contrast to
these r-strategists, species found in more stable situations face competition
for space and limited resources (Begon et al. 1986). These organisms, termed K-strategists,
become more dominant as a community matures and becomes more crowded. These
concepts form the endpoints of a continuum, and there are varying degrees of
r- and K-related characteristics in different microbes in various habitats
(see Andrews and Harris [19851 for further discussion on these concepts in
microbial ecology). Plant pathogens
are spread across this r-K range of characteristics and vary in other
important biological characteristics (Van Driesche & Bellows 1996). There
are opportunistic pathogens that are able to attack young, weakened, or
predisposed plants, but may be poor competitors (Botrytis, Pythium,
Rbizoctonia). There are pathogens that tolerate environmental stresses.
These organisms often live in situations with few competitors, because few
species are able to exist in such environments. Some pathogens, such as the Penicillium
species that cause postharvest rots, produce antibiotics that inhibit
competitors. Other species (such as Fusarium culmorum [Smith]
Saccardo) have a very high competitive ability. It is important to understand
the ecology of a target pathogen before one can effectively consider what
biological control strategy might be most effective. Stress-tolerant and
competitive species, for example,require different biological control
strategies and agents than ruderal ones. Similar to the way that antagonists of
plant pathogens vary in r-K and other characteristics, the properties of an
effective biological control agent will depend on the setting in which it is
intended to function, in many agricultural settings, disturbance makes new
resources available to microbes through crop residue burial, cultivation, or
planting. A frequent need, therefore, is a control agent that has the
characteristics of an r-strategist (Campbell 1989), which can grow quickly
and colonize new resources rapidly, with minimal nutrient and environmental
restrictions. It should function well in disturbed environments and have some
means (such as spores) of surviving in the soil or on the plant near to the
pathogen inoculum or the Source or site of infection. Biological control
agents that are r-strategists are an approximate equivalent of a protectant
fungicide, being in place before the pathogen infection cycle can begin. In
other programs, such as those directed against a pathogen which has already invaded
the plant host, a more competitive species will be required. Finally, a
biological control agent may have to be tolerant of abiotic stresses,
particularly for use in dry climates or on leaves. Although there
is much variation in soil types in different locations, soils are typically
rich in microflora, with propagules numbering in the hundreds of thousands
per gram of soil (Campbell 1989). In most soils, growth of microorganisms is
carbon-limited. either because what carbon is available is not physically
accessible or because the microbes do not possess the enzymes necessary to
degrade the carbon-containing molecules that are present. An exception to
this general limitation is the region immediately surrounding plant roots.
This region, the rhizosphere, contains easily metabolized carbon and nitrogen
sources such as amino acids, simple sugars, and other compounds exuded by the
roots. Consequently, this region is more favorable than surrounding soil for
the support of microflora. Root pathogens and plantparasitic nematodes may be
found growing on or in roots, but many microbes in the soil will be dormant
because of resource limitations. Because there are many dormant organisms in
the soil prepared to take advantage of any favorable period or opportunity,
competition for resources in the soil may be significant and may limit the
ability to augment beneficial organisms and have them flourish, unless soils
are first sterilized to eliminate potential competitors. Therefore, much
research surrounding biological control of root diseases and nematodes has
centered around identifying soils which are naturally suppressive to
particular disease organisms and investigating the microbial components of
the soil responsible for the suppression. Management of such antagonistic
organisms for biological control can range from treatment of soil to favor
the desirable organisms (conservation) through inoculation of soils or plants
with specific beneficial microorganisms (augmentation) (Van Driesche &
Bellows 1996). The phyllosphere
is significantly different from the rhizosphere in its structure, ecology,
nutrient availability, and exposure to climatic factors (Andrews 1992).
Leaves are relatively hostile to microorganisms. They are generally
hydrophobic and covered with chitin and wax, which limits the amount of
exudate (and hence nutrients) that reaches the leaf surface. These and other
factors impose severe environmental restrictions to microbial growth on leaf
surfaces. Fungal pathogens of leaves often enter the leaf tissue very shortly
after germination of the pathogen and, consequently, are protected inside the
plant for much of their growth. Bacterial pathogens may multiply on the leaf
surface before invading leaf tissues. Biological control of disease can take
place either through general inhibition and competition on the leaf surface
prior to invasion of leaf tissues or through suppression of the disease after
the pathogen has invaded. Biological control within leaf tissues can occur
through one of several mechanisms, including induced resistance in the plant
and hyperparasitism of the pathogen. Woody stems are habitats low in
nutrients and often difficult for pathogens to penetrate, Because the wood
itself supports very few saprotrophic microorganisms, pathogens colonizing
the wood through wounds, dead branches, or roots find very few competitors.
Because there are few organisms present to conserve, protection of the wood
from these decay organisms can be achieved by protecting the relatively
small, well defined wound or branch stub through inoculation (augmentation)
with specific microorganisms. These wounds are initially very low in sugars
or other nonstmctural carbohydrates, and antagonists such as Trichoderma spp.
can successfully compete for these limited resources. Many of the organisms
used in the biological control of stem diseases are employed by applying them
directly to stem wounds, where they colonize resources and subsequently exclude
pathogenic forms. This initial occupancy by antagonists subsequently limits
infection by decay-causing organisms, and hence controls the succession of
microorganisms in the wood. Of the successful, commercially-available
biological control products for plant diseases, several are for diseases of
woody stems (Campbell 1989). Plant Pathogen Biocontrol Mechanisms There are
several different ways in which a microbial biological control agent can
operate against a targeted plant pathogen (Elad 1986). Among these are
competition, induction of plant defenses, and parasitism. Some agents act
through competition for limited resources, and through this competition the
growth of the pathogen population is suppressed, reducing the incidence or
severity of disease. One important component of competition can be
competition for Fe-31 ions. These ions are sequestered by chemicals called
siderophores, which are produced by many species of plants and microbes.
Highly efficient siderophores from nonpathogenic microbes can remove Fe-3l
ions from the soil, outcompeting siderophores from pathogens and thereby
limiting the growth of pathogen populations. Some biological control agents
compete through the production of antimicrobial substances such as antibiotics
which inhibit the growth of pathogens directly, rather than by preemptive
consumption of limiting resources. An important
mechanism limiting infection is the induction of plant defenses against
pathogensby nonpathogenic organisms. Cross-protection and induced resistance
are mechanisms in which plants are intentionally exposed to certain
(nonpathogenic or mildly pathogenic) microbes, thereby conferring in the
treated plants some resistance to infection by pathogens. induced plant
defenses may include lignification of cell walls through the addition of
chemical cross-linkages in cell wall peptides which makes the establishment
of infection through lysis more difficult, suberification of tissues (where
plant cell walls are infiltrated with the fatty substance suberin, making
them more corklike), and other general defenses, including production of
chitinases and Beta 1,3-glucanases. These plant defenses then limit later
infection by pathogens, The biological control agent employed may be an
avirulant strain of the pathogen, a different forma specialis, or even
a different species of microorganism. A third
mechanism by which beneficial microorganisms suppress plant pathogens is
parasitism. Some species of Tricboderma, for example, attack pathogenic
fungi, leading to the lysis of the pathogen. Natural enemies of
plant-parasitic nematodes include bacterial diseases and nematophagous and
nematopathogenic fungi. As is the case
of conservation of natural enemies of pest arthropods and weedy plants,
conservation activities for the suppression of plant pathogens consist of
either avoiding practices which reduce desirable antagonists or actively
modifying the environment to favor or selectively enhance the growth of such
species. in the case of soil microflora, species employed for biological
control of plant pathogens are often competitive antagonists. Adding
amendments to soil is one way in which soil microorganisms may be managed to
enhance populations of these beneficial organisms. Addition of organic matter
to soils for control of Streptomyces scabies, the causative organism
of potato scab, is one example. Addition of carbon sources to soil increases
general microbial activity which leads to reductions in S. scabies. Specifically,
Bacillus subtilis and saprotrophic species of Streptomyces were
encouraged by barley, alfalfa, or soy meal (Campbell 1989). Soy meal was also
a substrate for antibiotic production against S. scabies. A general
rise in soil organic matter also gave control of Phytophthora cinnamomi Rands
in avocado in Australia (Manajczuk 1979). The addition of more than 10 tons
of organic matter per hectare per year led to general increases in numbers of
bacteria. Lysis of the hyphae and sporangia of the pathogen were attributed
to species of Pseudomonas, Bacillus, and Streptomyces. Some soils
appear to suppress disease naturally and may contain antagonistic or
antibiotic flora which flourish without the need for amendments. An example
of such suppressive soils is the Fusarium-suppressive soil in the
Chateaurenard District of the Rhone Valley in France Here, Fusarium
oxysporum f: sp. melonis Snyder and Hansen is present, but no disease
develops when susceptible melon varieties are grown. These soils are
suppressive for several other types of F. oxysporum, but not to other
species or genera of pathogens. The suppressive nature of the soils is
clearly biotic, because the soils lose their suppressive ability when
steam-sterilized, and the suppressive ability can be transfered to other
soils. The antagonists principally responsible for this suppression are
nonpathogenic strains of F oxysporum and F. solani (Martius)
Saccardo. The suppression appears to be due to fungistasis induced by
nutrient limitation. The competing fungi appear to have nearly the same
ecological niche as the pathogenic forms, and the saprotrophic forms
outcompete the pathogens for limiting resources so that dormant
chlamydospores of the pathogen do not germinate in the presence of host root
exudates. It may be possible to develop systems for other areas using the
antagonists from the Chateaurenard area (Campbell 1989), although additional
research may be necessary to permit their effective operation in different
soils. Other soils suppressive to Fusarium wilts are known. There are
numerous other examples of suppressive soils, although some soils or
combinations appear to give somewhat variable results (Van Driesche &
Bellows 1996). The conservation
of existing flora may be important in limiting the extent of a number of leaf
diseases (Campbell 1989). These effects are often revealed through the use of
fungicides which deplete extant fungi, permitting the development of
previously unimportant diseases, Fokkerna and de Nooij (1981), for example.
evaluated the effects of various fungicides on leaf surface saprotrophs that
have been used in biological control. Wide-spectrum fungicides allowed almost
no growth of saprotrophs, while more selective agents permitted some growth
of several genera of saprotrophs. in cases where these saprotroph populations
play an important role in limiting disease organisms, the application of
fungicides would eliminate their contribution to pathogen suppression. A case
illustrated by Fokkerrrt and de Nooij (1981), is where plants treated with
benomyl (a systemic fungicide) had fewer saprotrophs and developed more
necrotic leaf area when inoculated with Cocbliobolus sativus (Ito and
Kuribayashi) Drechsler ex Dastur than nontreatect plants (C. sativus is
insensitive to benomyl), Another example (Mulinge & Griffiths 1974) is
leaf rust of coffee (Coffea arabica L.), caused by Hemileia
vastatrix Berkeley and Broome. The disease can be controlled by proper application
of fungicide. However, if fungicides are applied in one year and not in the
next, the disease is worse on the treated plants than on those which did not
receive treatments either year. The elimination of the saprotrophic flora by
the fungicide removes their natural suppressing influence on the disease
organisms, permitting the disease to worsen , Here, careful use of selective
fungicides are crucial to conserving the important antagonistic flora and
permitting their beneficial action (Van Driesche & Bellows 1996). Several reports
exist of substantial natural control (control by natural enemies without
intentional manipulation) of plant-parasitic nematodes. Stirling (1991) and
Sayre and Walter (1991) review several of these; one example is that of the
natural suppression of the cereal cyst nematode Heterodera avenae in
cereal cultivation in the Great Britain (Gair et al 1969). In this case,
populations of the nematode initially increased for the first 2-3 years of
cultivations, and then declined continually during 13 years of continuous
cultivation of both oats and barley (a more susceptible crop). Four species
of nematophagous fungi were present in the soil. The two species principally
responsible for nematode suppression were Nematopbtbora gynophila and
Verticillium chlamydosporium. Both fungi attacked female nematodes,
either destroying them or reducing their fecundity. The activity of both
fungi was greatest in wet soils during laboratory trials (Kerry et al. 1980).
Although natural suppression of the nematode population required some time to
develop in these soils, but once established it maintained the population
below the economic threshold (Stirling 1991). Conserving
nematode antagonists in soils (as opposed to directly enhancing their
numbers), is a matter that has received relatively little attention (Van
Driesche & Bellows 1996). The application of toxins (insecticides,
fungicides) to aerial portions of crops or directly to soils often leads to
pesticide activity in the soil. All nematicides are nonselective in their
action and, hence, will kill predatory nematodes (Stirling 1991). In
addition, herbicides have well-documented effects on soil microorganisms
(Anderson 1978) and may well exert some influence on microbial antagonists of
nematodes, and insecticides may negatively affect soil microarthropods. Many
fungicides are known to be detrimental to nematophagous fungi (Mankau 1968;
Canto-Saenz and Kaltenbach 1984; Jaffee and McInnis 1990), but at levels
higher than would be expected under normal field practice. Among the fumigant
nematicides, ethylene dibromide (EDB) and eibromo-chloro-propene (DBCP)
appear nontoxic to the nematode-trapping fungi (Mankau 1968), and several
herbicides were shown to be unharmful to Arthrobotrys sp. (Cayrol
1983). Despite these potentially significant effects on beneficial microflora
and fauna and the possibility of conserving these organisms by appropriate
choice of material, little has emerged to integrate these ideas into normal
farming practice (Van Driesche & Bellows 1996). Perhaps because there has
been no serious emergence of nematode problems associated with the use of
these materials, this status quo is justified. Nonetheless, the
opportunities for conserving biologically important agents should be
considered in the development of future integrated management programs for
plant-parasitic nematodes. Similarly, cultivation practices may be selected
to favor natural enemies of nematodes, Among these are minimum or
conservation tillage, which reduced the number of cysts of Heteroderci
avenae on roots and the amount of damage caused by the nematode on wheat
in Australia (Roger and Rovira 1987). other practices which may affect
populations of natural enemies include normal tillage (which adds crop
residue to the soil and thus may favor certain beneficial organisms) and crop
rotation sequences (Stirling 1991). The knowledge that some soils are
naturally suppressive to nematodes prompts the question of whether or not the
features of these soils can be used to improve biological control. in all
documented instances where they have been studied, the suppressive properties
of these soils appear to result primarily from the action of one or two
specific biological control agents (Stirling 1991). The suppressiveness
requires substantial time to develop, and considerable crop loss might be
incurred during such an initial phase. Some risk is involved also, because
the suppressive nature of the soil may not develop to suitable levels.
Careful management of crop varieties, particularly using varieties resistant
or tolerant to nematode damage during the initial phases of land use for
cropping, is an important part of taking advantage of the potential of these
resident natural enemies. Agriculturists have large amounts of capital
invested in land, equipment, and cropping costs, and consequently require a
certain degree of reliability in pest control measures. Because of the
variable nature of natural suppressiveness of nematodes, any natural control
of nematodes in the foreseeable future is most likely to arise fortuitously
rather than result from any deliberate actions by scientists or farmers
(Stirling 1991). Where soils are not naturally suppressive to nematode
populations, they may be manipulated to enhance what natural control agents
are present. Most attention in this arena has been given to the addition of
organic matter to the soils. Much of the information regarding the effects of
these amendments is circumstantial, but the beneficial effects appear
widespread. Many different soil amendments have been considered and
evaluated, and the reduction of plant damage from nematodes following such
amendments may occur through a variety of mechanisms (Stirling 1991). One way is
through the general improvement of soil structure and fertility. Addition of
crop residue or animal manures increases ion exchange capacity of the soil,
chelates micronutrients to make them accessible by the plant, and adds
available nitrogen. Grown under such improved conditions, plants are better
able to tolerate damage from nematodes. Certain amendments may directly
improve plant resistance to nematodes (Sitaramaiah and Singh 1974). Others
may contain or release compounds which adversely affect nematodes. Among
amendments containing such compounds are those of neem (Azidiracbta indica
A. jussien) seeds or leaves and of castorbean (Ricinus communis L.)
(Stirling 1991 and references therein). Other amendments release nematicidal
compounds during decomposition. The most widely studied of these compounds is
ammonia. Because nitrogen is a constituent of nearly all soil amendments,
ammonia is usually produced during decomposition. A careful lialance must be
maintained in the carbon:nitrogen ratio, together with sufficient
concentrations of ammonia, to provide optimal effect without phytotoxicity
(Stirling 1991). Finally, there
is the direct stimulation of nematophagous or antagonistic organisms. Spores
of many nematophagous fungi fail to germinate in otherwise suitable but
nonamended soils (Dobbs and Hinson 1953), and this soil mycostasis can affect
both spores and mycelia (Duddington et al. 1956ab, 1961; Cooke and
Satchuthananthavale 1968). Before predation of nematodes can take place,
mycelial growth and trap formation must occur. The addition of organic matter
provides a substrate which may stimulate spore germination. Organic
amendments stimulate a broad range of soil microorganisms, so the effects of
amendments on populations of these organisms is complex. Microbial population
growth generally increases immediately following the addition of organic
matter and, subsequently, as pan of the community succession, there is an
increase in populations of nematode-trapping fungi. The general hypotheses regarding
the beneficial effects of organic amendments center around the stimulation of
the saprotrophic growth phase of nematophagous fungi, and stimulation of
other general microorganisms which may be detrimental to nematodes, such as
antibiotic producing bacteria. A general rise in enzymatic levels also occurs
following soil amendment, and the enzymes may attack the structural proteins
in nematode cuticle or egg shell. Chitin amendments in particular have
received attention, and addition of chitin to soil is followed by a
relatively long-term (4-10 weeks) rise in chitinase activity in the soil.
Chitin is the principal structural component of nematode egg shells, and the
increase in chitinase activity may be accompanied by decreased survival of
nematode eggs. However, the decomposition of chitin also releases ammonia,
which may contribute to its beneficial effects. Speigel et al. (1988, 1989)
concluded that the beneficial effects of chitin amendments resulted from the
action of specialized microorganisms. A current limitation of the
implementation of amendments for nematode control is that such amendments
must be applied in large amounts, between 1-10 tons/ha to be effective. The
use of local resources for such amendments will keep transport costs minimal.
One product, the chitin-based Clandosant (derived from crab shells), has been
marketed commercially. There is some evidence that the effectiveness of
certain amendments may be enhanced by inoculating them with degradative
microorganisms (Galper et al. 1991), and Stirling (1991) suggests
consideration of systems in which amendments can be inoctitated with a
specific microorganism as they are applied to the soil. Augmentation of
antagonists of plant disease organisms can generally be of two types,
inoculation and inundation. Inoculative releases consist of small amounts of
inoculum, with the intention that the organisms in this inoculum will
establish populations of the antagonist which will then increase and limit
the pathogen population, In inundative releases, where large amount of
inoculum is applied, with the expectation that control will result directly
from this large initial population with limited reliance on subsequent
population growth. Biological control of plant pathogens may also rely on a
hybrid of these two concepts. A large amount of inoculum must be applied,
both to increase the population of the antagonist and to improve its
distribution to favor biological control. Also, antagonism can result from
both these applied organisms and the increased population of antagonists
resulting from their reproduction. Biological control of blackcrut (Phyllachora
huberi) on rubber tree foliage by the hyperparasites Cylindrosporium
concentricum and Diicyma pulvinata (Junqueira and Gasparotto 1991)
is one example of long-term control of a plant pathogen by a single
augmentation in an agricultural system (Cook 1993) In this case, rubber trees
were treated with spore suspensions of the antagonists (inundatively), which
resulted in control over more than one season. More generally, beneficial
microorganisms are added seasonally or more frequently. Where the beneficial organisms involved are being placed into a
habitat or environment other than where they originated, the organisms are
often referred to as "introduced" in plant pathology (Andrews 1992;
Cook 1993). There are several examples where such organisms, when moved to a
new habitat (for instance, from the soil to the above-ground part of a plant)
colonize and serve as successful agents of biological control (Andrews 1992;
Cook 1993). Competitive Antagonists &
Antibiotic Production
Root Diseases. One way in which flora may be manipulated to protect
against disease is to intentionally inoculate soils or seeds with microbial
antagonists. Such antagonists, to be successful in their task, must be able
to colonize plant surfaces and survive in the competitive environment of the
soil. Flora with demonstrated ability to achieve this under field conditions
include fungi, principally Trichoderma spp., and, among the bacteria,
Bacillus spp. and Pseudomonas spp. Among the
bacteria, species of Bacillus are regularly used for biological
control of root diseases, Members of the genus have advantages, particularly
that they form spores which permit simple storage and long shelf life, and
they are relatively easy to inoculate into the soil. However, the consequence
of this biology is that although the inoculant may be present in the soil, it
may be in dormant or resting stages. Nonetheless, species of Bacillus have
provided good control on some occasions. Capper and Campbell (1986) showed a
doubling of wheat yield over wheat plants naturally infected with take-all by
those also inoculated with Bacillus pumil Meyer and Gottheil. Bacillus
pumilus and B. Subtilis were also used to protect wheat from
diseases caused by species of Rhizoctonia (Merriman et al. 1974). A
major difficulty with the use of Bacillus spp. is that the control provided
is often variable, with different results in different locations. or even in
different parts of a season in the same location (Campbell 1989). Bacillus
subtilus is used as a seed inoculant on cotton and peanut (Arachis
hypogaea L.) with nearly 2 million ha. treated in 1994 (Blackman et al.
1994). Treatment promotes increased root mass, modulation, and early
emergence, and suppresses diseases caused by species of Rhizoctonia and
Fusarium. Of much more
promise as antagonists of root diseases are species of Pseudomonas,
particularly the Pseudomanas fluoresce and Pseudomonas putida
(Trevisan) Migula groups (Campbell 1989). These bacteria are easy to grow in
the laboratory, are normal inhabitants of the soil, and colonize and grow
well when inoculated artificially. They produce a number of antibiotics as
well as siderophores. Several have received patents and are marketed
commercially for control of root rot in cotton (Campbell 1989). An isolate of
another species of Pseudomonas has been used as anantagonist of
take-all disease of wheat (Welterl983). Isolates of Ps.fluorescens
from soils showing some control of take-all can be applied as seed coats and
inoculated into fields suffering from the disease. Such treatments give
10-27% yield increases compared with untreated, infected control groups.
Evidence points to both siderophore and antibiotic production as important. Species of the
fungal genus Trichoderma can be saprotrophic and mycoparasitic and
have been used against wilt diseases of tomato, melon, cotton, wheat, and
chrysanthemums. The antagonists were applied to seeds or through a bran
mixture incorporated into the planting mix at transplanting. Although disease
did develop, it did so much more slowly than in untreated soils, resulting in
a 60-83% reduction in disease (Siven and Chet 1986). The mode of action
against Verticillium albo-atrum Reinke and Berthier wilt of tomatoes
appeared to be antibiosis. Stem Diseases. The control of Heterobasidion
annosum, the causative agent of butt rot in conifer stumps, by Pbanerochaete
gigantea was one of the first commercially available agents for
biological control of a plant pathogen (Campbell 1989). The disease caused by
H. annosum is primarily a disease of managed plantations. The
fungus colonizes freshly cut stumps, invades the dying root system. and can
then infect nearby trees through natural root grafts, causing death of the
trees. However, Heterobasidion annosium, is a poor competitor, and
when a stump is intentionally inoculated with Ph. gigantea (and
usually with chemical nitrogen sources which encourage growth of the
antagonist) the antagonist rapidly colonizes the resource, excluding future
attack by the pathogen and even eliminating existing pathogen infection
(Table 12.1). Very little inoculum is needed on a freshly-cut stump, and the
shelf life of the pellet formulation is about two months at 22'C. The
antagonist is able to outcompete H. annosum even when the initial
inoculum favors the pathogen by as much as 15:1 (Rishbeth 1963). The ascomycete
fungi Eutypa armeniaceae Hansford and Carter and Nectria galligena Bresadola
& Strass infect apricots and apples, respectively, and cause stem cankers
and eventual death of the trees. Pruning wounds in apricots are treated with Fusarium
laterium Nees:Fries through specially adapted pruning cutters. Fusarium
laterium produces an antibiotic which inhibits germination and growth of E.
armeniaceae. When applied, the concentration of the antagonist must be
greater than106 conidia/ml. Integrated application which includes a
benzimidazole fungicide gives better control than either fungicide or
antagonist alone. Nectria galligena infection can be reduced through
sprays of suspensions of Bacillus subtilis or of Cladosporium
cladosporioides (Fresenius) de Vries. These antagonists are not in
commercial use because apples are treated for Venturia inaequalis (Cooke)
G. Winter (apple scab) so frequently that V. galligena is controlled
by those sprays. Crown gall is a
stem dlisease caused by the bacterim Agrobacterium tumefaciens (Smith
& Townsend) Conn. It affects both woody and herbaceous plants in 93
families. Infection is typically from the soil, rhizosphere, or pruning
tools. Control can be effected by treating plants with a suspension of a
related saprotrophic bacterium Agrobacterium radiobacter (Beijerink
and van Delden) Conn strain K-84. This strain of the bacterium produces
an antibiotic which is taken up by a specific transport system in the
pathogen bacterium, which is then killed. The commercially available
formulations of this agent are effective primarily against pathogen strains
which attack stone fruits, but other bacteria are under investigation for use
against strains pathogenic in other crops. This agent has been altered by
gene-modifying technology to produce a new strain (strain 1024) which lacks
the ability to transfer antibiotic resistance to the target bacterium (Van
Driesche & Bellows 1996). The fungus Cbondrostereum
purpureum (Persoon: Fries) Pouzar infects stems of fruit trees and
produces a toxin which leads to a condition known as silverleaf disease.
Stems can be inoculated with a species of Trichoderma grown on wooden
dowels or prepared as pellets which are inserted into holes bored in the
affected stem. Treated stems recover from the disease more rapidly than
untreated stems. The Trichoderma sp. can be applied to pruning wounds
to prevent initial establishment of C. purpureum. Leaf Diseases. Control of leaf diseases at the time of pathogen
germination has been demonstrated in the laboratory. This control occurs in
the presence of competitive organisms, which may include fungi, yeast, or
bacteria. The mode of action in some cases is competition for nutrients
which, together with water, are necessary for successful germination and
invasion of many pathogens. The germination of Botrytis sp., for
example, is inhibited by certain bacteria and yeasts (Blakeman and Brodie
1977). This inhibition is less pronounced if additional nutrients are
supplied, indicating that the mechanism is, at least in parrt, resource
competition. Studies on control of Botrytis rot in lettuce (Wood 1951)
indicated that several organisms were successful in suppressing the disease
when sprayed on lettuce plants, among them species of Pseudomonas,
Streptomyces, Ttichoderma viride, and Fusarium. Peng and Sutton (1991)
evaluated 230 isolates of mycelial fungi, yeasts, and bacteria and tested
them as anntagonists of B. cinerea in strawberry in both laboratory
and field trials. Several organisms (including members of each taxonomic
group tested) were effective, some as effective as captan (a commercial
fungicide). Sutton and Peng (1993b) further evaluated Gliocladium roseum and
determined that the suppression of B. cinera by this antagonist was
probably a result of competition for leaf substrate. The fungi Gliocladium
roseum and Myrothecium verrucaria (Albertini and Schweinitz)
Ditmar were also effective in suppressing B. cinerea in black spruce (Picea
mariana [Miller] Britton Stearns Poggenburg) seedlings (Zhang et al.
1994). Bacteria may
also be used to limit frost damage to leaves and blossoms of plants. Certain
bacterial species such as Pseudomonas syringae and Erwinia
herbicola serve as nucleation sites on leaves for the formation of ice,
and, in their presence, ice forms soon after temperatures fall below
freezing. If these ice nucleating bacteria are replaced by competitive
antagonists (such as certain strains of Ps. syringae) that lack the
protein that causes ice nucleation, frost is prevented even at temperatures
from -2 to 5E C.(1 (Lindow
1985b). The protective bacteria, after being applied it) the leaves, colonize
them for up to two months, an interval suitable to protect from frost during
the limited season that low temperatures are likely. A naturally-occurring,
non-ice nucleating strain of Ps. fluorescens is registered in the
United States as a commercial product (Frostban B ) for suppression of frost
damage (Wilson and Lindow 1994). Spraying
suspensions of propagules, generally at high concentrations, is the principal
method for applying biological control agents to foliage (and to flowers),
and dusts (such as lyophilized bacterial preparations) are also used. Spray
methodology has yet to be refined in terms of sprayer characteristics,
droplet size, and pressures, and other methods of application with greater
efficiency may be necessary to effectively target certain plant parts (Sutton
& Peng 1993a). Flower Diseases. A principal disease of flowers which has received
attention is fire blight of rosaceous plants, which is particularly severe on
pear (Campbell 1989). The causal bacterium, Erwinia amylovora, also
occurs on leaves and may cause stem cankers. The bacterium is transferred by
insects to flowers in the spring from overwintering sites on stem cankers,
and subsequently from flower to flower. Infection enters the pedicel and from
there the stem. Infected flowers and small stems die, and cankers form on
other stems. Chemical control is difficult and expensive, and sometimes is
ineffective because of resistance to copper compounds and streptomycin.
Biological control has been effective using E inia berbicola, sometimes
in combination with Pseudomonas syringae (Wilson and Lindow 1993).
Suspensions of E. berbicola are sprayed onto the flowers just before
the period of potential infection. The antagonist occupies the same niche as
the pathogen, reducing the numbers of E. amylovora by competition, and
there is also evidence for the production of bacteriocins (chemicals which
suppress population growth of related bacteria) by some strains. Control can
be good, comparable to that achieved by commercial bactericides, though
repeated application of the bacterium was necessary (Isenbeck and Schultz
1986). Another approach to control is to reduce secondary infections on
leaves, which leads to reductions in the overwintering population of the
pathogen. This control is achieved by treatment with the antagonists
Ps.syringae and other bacteria (Lindow 1985b). A novel approach to
dissemination of the antagonistic bacteria has been evatuated by Thomson et
al. (1992). They mixed E. herbicolaancl Ps. fluorescens with pollen in
a special apparatus at the entrance to honey bee (Apis mellifera) hives.
Bees emerging from these hives through the mixtures transmittecl the
antagonists to the flowers efficiently, although disease control was not
evaluated because of absence of disease in the test orchards. Fruit Diseases. Fruits are subject to attack both by general pathogens (Botrytis,
Rhizopus, Penicillium) and by a few specialist pathogens such as the
coffee berry disease fungus Colletottichum coffeanum Noack and Monilinia
spp., which cause brown rots of rosaceous fruits. While many of these are
controlled by fungicides, Trichoderma viride has been shown to limit
disease from Monilinia spp. Various Bacillus spp. also are
antagonistic to these fungi through production of antibiotics and by reducing
the longevity and germination of spores. Both the bacteria and culture
filtrates have been used with some success against these pathogenic fungi,
but there has been no commercial development, probably because fungicides
used routinely in orchards for control of other diseases give some control of
brown rot (Campbell 1989). Among the most serious diseases of soft fruits are
postharvest rots (Dennis 1983), especially that caused by Botrytis
cinerea. Potential for biological control of postharvest diseases was
reviewed by Wilson and Wisniewski (1989) and Jeffries & Jeger (1990)
(also see Wilson and Wisniewski 1994). In strawberries, B. cinerea grows
saprotrophically on crop debris and from there infects flowers or fruit.
Various species of Trichoderma have been evaluated and gave control as
good as standard fungicides (Tronsmo and Dennis 1977). The antagonists Clado,@Cladosporium
herbarium and Penicillium sp. gave excellent results in
controlling Botrytis rot on tomato (Newhook 1957). Honey bees have
been used to distribute Gliocladium roseum to strawberry flowers (Peng
et al. 1992) and raspberry flowers (Sutton and Peng 1993a) to suppress Bot?ytis
rot. Root Diseases. Induced resistance is a form of biological control in
which the natural defense responses of the plant, which may include
production of phytoalexins, additional lignification of cells, and other
mechanisms (Horsfall and Cowling 1980; Bailey 1985), are promoted in the
plant prior to exposure to the pathogen (Van Driesche & Bellows 1996).
These resistance mechanisms are induced by challenging the plant with a
nonpathogenic organism. The induced plant defenses then limit later infection
by the pathogen. The organism employed may be an avirulent strain of the
pathogen, or a different specialized form, or even a different species, There
are few well-documented cases of induced resistance for soil-borne pathogens,
and these are mostly of wilt diseases. Dipping tomato roots in a suspension
of Fusarium oxysporum f.sp. dianthi a few days before likely
exposure to the pathogen F oxysporum f.sp. lycopersici (Saccardo)
Snyder and Hansen conferred protection that lasted a few weeks. Cotton may be
protected for three months or longer by spraying the roots at transplanting
with a mildly pathogenic strain of the disease causing pathogen Vcrticillium
albo-atryum. The role of some fungi against take-all of wheat includes
some elements of induced resistance. Gaeumannomyces graminis var. graminis
grows on grass roots and also has been found on wheat, where it occupies a
niche similar to that of the pathogen G. graminis var. tritici Walker.
The antagonist invades the root cortex but not the stele, and is halted by
the lignification and suberization of the cortex and stele. Root cells with
these chemically-changed walls are less susceptilile to invasion by the
pathogen. Although this interaction produced yield increases in Europe, the
strains or species present in the United States did not appear to confer
resistance, and in Australia there were only slight yield increases (Campbell
1989). These variable results, while somewhat common for biological control
of soil borne pathogens, do not reduce the value of the antagonists where
they do work, but rather indicates some potential challenges in defining the
taxonomy, biology, and host-plant relationships important to biological
control in this group of organisms. Leaf and Stem
Diseases. Induced resistance can control anthracnose diseases caused
ny Colletotrichum spp. (Kúc 1981; Dean and Kúc 1986). Colletotricbum
lindemthianum (Saccardo & Magnus) Lamson Scribner causes anthracnose
of beans, Colletotrichum lagenarium (Passerine) Saccardo causes
cucumber anthracnose, and Cladosporium cucumerinum Ellis and
Arthur causes scab in cucumbers. Inoculation of cucumbers with Colletotrichum
lindemuthianum (which does not cause disease in cucumbers) made plants
resistant to both Colletotrichum lagenarium and Cladosporium
cucumerinum. Treatment applied to an early leaf resulted in protection of
later leaves, even when the initially inoculated leaf was removed. The factor
causing resistance travels systemically through the plant. Variations on this
approach include inoculating an early leaf with a pathogen, inducing
resistance throughout the plant, and then removing the infected leaf. Induced
resistance also occurs in some virus diseases (Thomson 1958) and may last for
years, as in the case of healthy citrus seedlings being inoculated with an
avirulent strain of citrus tristeza vinus Stem rot in
carnations, caused by Fusarium roseum Link: Fries 'Avenaceum,' can
be prevented by inoculating wounds inflicted during propagation with the
nonpathogenic F. roseum 'Gibbosum.' This inoculation produced a
germination inhibitor and also reduced the time needed for the stems to
develop resistance to the pathogen. This hastening of resistance was caused
by activation of the host's defense mechanisms, and is another example of
induced resistance. Chestnut blight,
caused by Cryphonectria parasitica, is controlled by employing
hypoviruient strains of the disease pathogen. A number of hypovirulent
strains are known, and inoculating infected trees with a hypovirulent strain
leads to reduced canker size and greater stem survival. In the field,
hypovirulent strains are inoculated into infected trees at the rate of 10
inoculated trees/ha. The hypovirulent strain spreads from these locations
and, on contacting more virulent strains, fuses with these strains and
exchanges a viral element infecting the pathogen (Van Driesche & Bellows
1996). The hypovims, which causes hypovirutence, is transferred to the
virulent strains, attenuating their effects. Active cankers are eliminated in
10 years (van Alfen 1982). Parasitism of Pathogens and Nematodes Root Diseases. The mycoparasites Tiichoderma spp, have been used
successfully against diseases caused by Rbizoctonia and Sclerotium pathogens.
One example is the pathogen Sclerotium rolfsiii Saccardo, which
attacks many crop plants and survives unfavorable periods by forming
sclerotia in the soil. Strains of T barzanium that have Beta 1-3 glucanases,
chitinases, and proteases have been isolated. These enzymes permit T barzanium
to parasitize the hyphae and sclerotia of the pathogen, invading and
causing lysis of the cells. Trichoderma harzanium is grown on
autoclaved bran or seed, and this material is then mixed with the surface
soil (Chet and Henis 1985). Two other fungi known to parasitize sclerotia are
Coniothryium minitans and Sporidesmium sclerotivorum (Ayers and
Adams 1981). Sporidesmium
sclerotivorum is a hyphomycete that in nature behaves as an obligate
parasite of sclerotia of Botrytis cinerea and several species of Sclerotium
(Adams 1990). It has been studied as an agent against botrytis rot in
lettuce, where it shows considerable potential. It can be grown in vitro on
various carbon sources and is efficient in converting glucose into mycelium.
Spores produced in mass culture are collected, processed, and applied to
infected soil, and field tests are promising (Adams 1990). Leaf Diseases. Some plant pathogens, including fungi and some bacteria,
are known to be attacked by other pathogens. Bdellovibrio bacterivorus is
a bacterium that can attack other bacteria by penetrating the cell wall and
lysing the host bacterium, subsequently reproducing inside its host.
Different strains of Bd. bacterivorus have been examined for virulence
against Pseudomonas syringae pv. glycinae (Coeper) Young, Dye and
Wilkie, the cause of soybean blight. By applying Bd. bacteriovorus at
sufficiently high rates, disease symptoms were reduced more then 95% (Scherff
1973). Parasites of fungi pathogenic on leaves are numerous (Kranz 1981), but
only a few have been studied in much detail, such as Sphaerellopis filum,
Verticillium lecanii and Ampelomyces quisqualis. The mycoparasite
typically penetrates the host hypha or spore and kills it. Some of the
control may be from the pathogen overgrowing the sporulating pustules of the
pathogen and preventing spore release and thus reducing inoculum in the
environment, even if the spores are not killed. A typical problem with
implementation of these mycoparasitic fungi is that they often do not affect
a large proportion of the pathogens unless humidity and temperature are high.
Consequently, although much reduction of spore production may take place,
there is still sufficient inoculum of the pathogen remaining to cause
disease. These mycoparasites often are seen only at high incidences of
disease, which is unsuitable for general control of the target pathogens.
They may have some use in particular systems, either in the tropics or in
greenhouses, where environmental conditions are more favorable. Plant-Parasitic Nematodes. The bacterial pathogen of nematodes most studied is Pasteuria penetrans sensti stricto Starr and Sayre (Starr and Sayre 1988), which is an obligate parasite of root-knot nematodes (Meloidogyne spp.) and has not been successfully cultured in vitro. This restriction in mass culturing has limited attempts to test the bacterium's effectiveness (Stirling 1991). In experimental trials, it has shown potential for controlling root-knot nematodes (Meloidogyne spp.) (Mankau 1972; Stirling et at. 1990), infesting a high proportion of nematodes in soil to which bacterial spores had been added, and in other trials (U. S. Department of Agriculture 1978) reducing damage to plants in plots containing the bacterium. Observations by Mankau (1975) indicated that populations of the bacterium did not increase rapidly in field soil. The development of a mass production method in which roots containing large numbers of infected Meloidogyne spp. females were air-dried and finely ground to produce an easily handled powder enabled more extensive testing (Stirling and Watchel 1980). When dried root preparations laden with bacterial spores were incorporated into field soil at rates of 212-600 mg/kg of soil, the number of juvenile Meloidogyne. javanica (Treub) Chitwood in the soil and the degree of galling was substantially reduced (Stirling 1984); other authors have reported similar results (Stirling 1991). Effective use of this bacterium through such inundative release would require concentrations on the order of 105 spores/g soil (Stirling et al. 1990). Such quantities could only be produced on a large scale with an efficient in vitro culturing method, a problem which has received attention but has not yet yielded a solution (Stirling 1991). Use in inoculative releases, where smaller numbers of spores are applied and a crop tolerant of nematode damage is grown to permit the increase of both nematode and bacterial populations, has been suggested (Stirling 1991). Conserving the bacterium in the presence of nematicides appears possible. Of seven tested nematicides, only one showed slight toxicity to the bacterium (U.S. Department of Agriculture 1978). The use of Bacillus thuringiensis strains with activity against nematodes is also possible. As these bacteria may be cultured in fermentation media. their mass culture is simpler than for P penetrans. |