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BIOLOGICAL
CONTROL OF PLANT PATHOGENS
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Competitive
Antagonists & Antibiotics Parasitism
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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 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 that 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. 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). 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 that attack pathogenic fungi (Kranz 1981). Among
those that 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). 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 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 were 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). 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 aboveground 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
that 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. Chemicals called siderophores, which are produced
by many species of plants and microbes, sequester these ions. 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 that 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 that may stimulate spore germination. Organic amendments
stimulate a broad range of soil microorganisms, so the effects of amendments
on populations of these organisms are 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 eggshells, 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 inoculants 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 that 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 that, 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. Insects transfer the bacterium 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). Challenging the plant with a
nonpathogenic organism induces these resistance mechanisms. 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: inoculating wounds
inflicted during propagation with the nonpathogenic F. roseum
'Gibbosum' can prevent Fries
'Avenaceum,'. 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. Employing
hypoviruient strains of the disease pathogen controls chestnut blight, caused
by Cryphonectria parasitica. 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. Suppression of
nematodes was possible through drench applications and through incorporating
the bacterium into a methyl cellulose seed coat (Zuckerman et al. 1993). Considerable
attention has been given to the nematode-trapping fungi as possible
augmentative agents, Mass culture on nutrient media is possible for these
fungi. Two cultures of nematophagous Arthrobotrys fungi have been
developed and tested for addition to soil for specific target environments.
Cayrol et al. (1978) reported the successful use of Arthrobotrys robusta Cooke
and Ellis var. antipolis, commercially formulated as Royal 300 against
the mycetophagous nematode Ditylenchus myceliophagus Goodey in
commercial production of the mushroom Agagaricus bisporus (Lange)
Singer. The nematophagous fungus was seeded simultaneously with A. bisporus
mushroom compost, which led to 280/o increases in harvest and reduced
nematode populations by 40%. The results justified the commercial use of the
fungus for nematode control in mushroom culture, Cayrol and Frankowski (1979)
reported the use of Arthrobotrys superba Corda (Royal 3509) in tomato
fields, applied to the soil at a rate of 140 g/M2, resulting in protection of
the tomatoes and colonization of the soil by the fungus. Other reports have
indicated little efficacy of fungal preparations when added alone to soil
(Barron 1977; Sayre 1980; Rhoades 1985). In general, there has been limited
success in the use of these agents (see Stirling [1991] for a summary). The
fungistatic nature of soil (Mankau 1962; Cooke and Satchuthananthavale1968)
may limit the ability of these fungi to grow even when added in substantial
numbers to soil. Additional work is needed, perhaps in the areas of
colonization and soil amendments together for the use of nematophagous fungi
to become suitably reliable for general use as a control method (van Driesche
& Bellows 1996). Many
predacious fungi may be unsuited for control of root-knot nematodes, Meloidogyne
spp. Stirling (1991) suggested that Monacrosporium lysipagum
(Drechsler) Subramanian and Monacrosporium ellipsosporum (Grove) Cooke
and Dickinson, which can invade egg masses, may warrant further
investigation. The nematode-trapping fungi are likely to be more effective
against ectoparasitic nematodes and such species as Tylenchus
semipenetrans Cobb, where juvenile stages migrate through the
rhizosphere. Little attention has been given to testing predacious fungi
against such nematodes (van Driesche & Bellows 1996). Fungi which
are internal parasites of nematodes are difficult culture on nutrient media,
and consequently there have been few attempts to use them for augmentative
control of nematodes. Alternative mass-culturing techniques may hold promise
(Stirling 1991). In the few experiments reported, the fungistatic effects of
soil often limited fungal growth and the effectiveness of the antagonists.
Lackey et al. (1993) report the production and formulation of Hirsutella
rhossiliensis Minter et Brady on alginate pellets (see also Fravel et al.
1985) which, when added to soil, led to transmission of the fungus to the
nematode Heterodera schachtii Schmidt and suppressed nematode invasion
of roots. Among the
facultatively parasitic fungi which attack nematodes, Paecilomyces
lilacinus and Verticillium cblamydosporium have received the most attention
as possible augmentative agents. The results of studies on P lilacinus have
been variable, with some studies showing some positive effect of the fungus,
while others show little or no effect (Stirling 1991). The mechanisms leading
to the beneficial effect have not been clearly elucidated, but may be from
metabolic products or effects other than direct parasitism of eggs. Studies
have generally involved the addition of fungal preparations to the soil at
the rate of 1-20 tons/ha, which is really too great for widespread commercial
use. Additions at lower rates (0.4 tons/ha) in a variety of carriers
(alginate pellets, diatomaceous earth, wheat granules) have also shown
limited beneficial effects (Cabanillas et al. 1989; Stirling 1991). Tribe
(1980) suggested the direct addition of V. Chlamydosproium to the
soil. Kerry (1988) added hyphae and conidia, formulated in sodium alginate
pellets or in wheat bran, to soil, and the fungus proliferated in the soil
only from granules containing bran. When chlamydospores were used as
inoculum, the fungus was able to establish without a food base (De Leij and
Kerry 1991). Of three isolates studied, only one successfully colonized
tomato root surfaces. This species apparently has considerable promise, but
screening programs will be necessary to identify isolates with
characteristics suitable for biological control (Stirling 1991). Predacious
microanhropods and nematodes have evoked considerable interest. Most work has
been done in simple microcosms, and there have been no attempts to evaluate
augmentative release of these organisms in a field setting. In one
experiment, Sharma (1971) found nematode numbers reduced by 50% or more in
glass jars inoculated with mites and springtails compared with similar jars containing
no predators, but the author pointed out possible causes for the reduction
other than simple predation. Experiments with predacious nematodes have in
general failed to demonstrate a measurable impact of the predator (Stirling
1991). One exception was the reduction of galling by Meloidogyne incognita
on tomato by predacious nematodes (Small 1979). The general suitability
of these groups of organisms for inundative release is questionable, because
of the potential difficulties in developing technologies for their rearing,
packaging, transport, and delivery beneath the soil in a viable state
(Stirling 1991). Mycorrhizae are nonpathogenic fungi
associated with roots in some temperate forest trees. Ectomycorrhizae are mostly
basidiomycetes which form a sheath over the root, and hyphae spread out into
the soil. These fungi have been studied in relation to nutrient uptake, but
they also affect root disease. Because they completely enclose the root, they
change the quantity and quality of exudates reaching the soil; consequently,
roots with mycorrhizae have a different rhizosphere flora than uninfectect
roots (Campbell 1985). In at least one case, the mycorrhizal fungus
Pisolitbus tinctorius (Persoon) Coker and Couch, the thick symbiont
sheath forms a barrier to infection by such pathogens as Phytophthora
cinnamomi attacking eucatyptus trees. Other mycorrhizal fungi produce
antibiotics effective against P cinnamomi in plate tests. The
intentional manipulation of mycorrhizal fungi for disease control has not
been widely implemented, but opportunities for selected uses may be possible
(Campbell 1989). Another group
of fungi are the vesicular arbuscular mycorrhizae (VAM), which are
phycomycete fungi associated with the roots of many plant species including
many crops. These fungi do not form a sheath surrounding the root, and their
effects on disease are complicated, but are in general beneficial (Campbell
1989). Some of these effects may involve changes in host plant physiology in
the presence of the symbiont, as there is no direct evidence of pathogen
inhibition by these fungi. Development
& Use of Beneficial Species Growth of our
knowledge about biological control of plant diseases has been extensive since
the first experimental reports (Hartley 1921; Sanford 1926; Millard,,ind
Taylor 1927; Henry 1931), and substantial potential for microbial control of
pathogens has been demonstrated. A number of products or programs have
reached the stage of commercial development or availability (van Driesche
& Bellows 1996). Products in current use include both those aimed at
specialty markets for control of certain stem or flower diseases (for which
chemical control is either unavailable or expensive) and those aimed at larger
scale markets such as seed treatments for widely planted crops. The cycle for
research, development, and implementation of antagonists of plant pathogens
is composed of several steps. These include initial discovery of candidate
agents, refinement of knowledge of their biology, ecology, and mode of
action, microcosm and field trials of their efficacy, and large-scale
development for commercial production. The first
challenge in the development of a biological control program is the discovery
process. Many microorganisms show potential as antagonists of particular
pathogens. Protocols have been proposed to make the process of screening
these candidates more efficient (Andrews 1992; Cook 1993). The principal
difficulties are screening out candidates that are effective only during in
vitro (agar plate) trials but are not effective in natural settings, and
in selecting candidates that can be successfully cultured in large
quantities. Following discovery of suitable candidates, research focuses on
their mode of action and on factors which may enhance or limit their efficacy
in targeted settings (glasshouses, field plots). In addition, experimental
fermentation and formulations must be developed for production of materials
suitable for use in agricultural settings. Finally, issues of large-scale
production and delivery must be addressed. Products for use must be effective
on an economical basis, and economies of scale may play an important role in
the eventual availability of any organism or product. Each must have a
satisfactory shelf life, and safe and effective methods for application must
be discovered or developed (Cook 1993; Sutton and Peng 1993a). Such
application methods might include sprays of suspensions or dusts, contact
application, honeybee and other bee vectoring, and production of antagonists
in a crop environment (Sutton and Peng 1993a). Adopting any
biological control agent in commercial agriculture is dependent on its
reliability and availability. Limitations to the process of eventual
adoption, therefore, include cost of development and size of potential market
(van Driesche & Bellows 1996). Many pesticides for control of plant
diseases have a broad spectrum of activity, are applicable in a variety of
crops and settings, and may act either prophylactically, therapeutically, or
both. Biological controls, in contrast, often have narrow ranges of activity
and may work in only a few crops or soil types, and while they can often act
both as a barrier and therapeutically, their action may take some time to
develop. Therefore, they may have a narrower market than a chemical pesticide
and be unattractive for development by major corporations (Andrews 1992). In
this context, it may be appropriate for public institutions such as
government experiment stations to undertake the development of such
biological controls, in the same way that they take the responsibility for
development of new plant varieties (Cook 1993). Those
microorganisms intended for use as biological control agents must be viewed
in a biological rather than a chemical control (Cook 1993). Where an
effective pesticide may work in many places, each place may have unique
edaphic, and biological features which limit or enhance the effectiveness of
microbial antagonists of pathogens. Consequently, each microbial biological
control system may have to make use of locally adapted strains, taking
advantage of resident antagonistic flora and fauna and augmenting their
effectiveness with additional species or strains, or enhancing resident
populations through soil amendments. Although the different strains may use
common mechanisms to achieve biological control (such as production of
antibiotics), competitive abilities adapted to local conditions may be vital
to permit the organisms to compete for resources and effectively control
pathogens (van Driesche & Bellows 1996) REFERENCES: <vandries.ref.htm> [Additional references may
be found at MELVYL Library ] |