CHAPTER 2

LITERATURE REVIEW

2.1 Biological control

Biological control means the reduction of inoculum density or disease-

producing activities of a pathogen or parasite in its active or dormant state. The

activity was performed by one or more organisms, accomplished naturally or through

manipulation of the environment, host, or antagonist, or by mass introduction of one

or more antagonists (Baker and Cook, 1974). The term biological control has been

used in this context to describe the use of living organisms or their products, to

combat the damaging activities of other organisms which are potential pests or

pathogens of plants. This was on the basic that pests have natural enemies and

biological control systems are designed to manipulate and enhance these phenomenon

in order to reduce the pest populations and to limit their activities (Isaac, 1992).

The objective can be achieved in a number of ways. For example, many

pathogenic fungi are poor competitors and may be quickly excluded from a site, such

as a leaf surface, if species which are more antagonistic are present. Some such

combative fungi are highly aggressive and produce toxic metabolites which quickly

affect less competitive individuals. Additionally, some fungal species are able to

parasitise and directly attack insects, nematode pests, or other fungal pathogens. The

use of various inoculation systems to encourange these interactions has been shown to

enhance the effectiveness of such natural biological control (Isaac, 1992).

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Biological control systems are preferred to the use of chemicals and in recent

years a great deal of research activity has been directed towards the development of

efficient and reliable systems. In environmental terms the effects and long term

consequences of biological control, are much less damaging than the routine use of

pesticides or fungicides. Treatments can be economic and cost-effective providing

that good control can be established, particularly when repeated chemical spraying is

required during the growing season. Biological systems have great potential in the

control of soil-borne microbes which are particularly difficult to treat by spraying

alone. Since the pest and antagonist are developing within a natural situation, there

will be co-evolution between them and the potential for the development of stable

resistance to the biological control agent is much reduced from that of chemical

treatments (Briese, 1986).

It is interesting that relatively few instances of biological control have been

effectively implemented in commercial field situations to date. Much research has led

to the development of efficient systems on laboratory or greenhouse scales where the

environment is highly controlled and predictable. However, once trials are scaled up

the extreme variability and unpredictability of natural field sites can lead to problems.

In theory, biological control, once established in a balanced situation, could be self-

perpetuating but in practice such systems are more often used as part of an integrated

control programme with pesticides and fungicides as supplementary treatments (Way,

1986; Burge, 1988) in much reduced quantities.

Example of plant protection by biological control including tomato, bell

pepper, celery and citrus. They were propagated in planting mixes amended with

formulations of commercial biocontrol agents. Root colonization by selected

biocontrol agents was evaluated for pepper, tomato and citrus, and found to be

generally between 76 to 100% in both greenhouse ebb and flow, and bench-produced

plants. All biological control agents, Trichoderma harzianum, Bacillus subtilis, G.

intraradices, Gliocladium virens, and Streptomyces griseovirdis reduced crown rot of

tomato in the field, with T. harzianum and B. subtilis being the most effective

uniformly among four tests. Four biocontrols reduced Phytophthora root rot on citrus

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in the field, two applied as a drench to soil in pots reduced Thietaviopsis root rot on

citrus, and two biocontrol agents in combination reduced celery root rot caused by

Pythium and Fusarium spp. (Nemec et. al., 1996). Six isolates of plant growth-

promoting fungi (PGPF), non-pathogenic Fusarium oxysporum, and five isolates of

bacteria were tested in hydroponic rock wool systems as potential biocontrol agents of

Fusarium crown and root rot (FCRR) of tomato caused by Fusarium oxysporum f. sp.

radicis-lycopersici (FORL) (Horinouchia et. al., 2006). In addition Arthrovacter spp.,

Azotobacter spp., Pseudomonas spp., and Bacillus spp. was used to control Fusarium

verticillioides in the maize rhizosphere. They were applied under greenhouse

conditions and it was found that Azotobacter armeniacus inhibited all F.

verticillioides strains assayed (Cavaglieri et. al., 2004).

2.1.1 Factors involved in biological control

Biological control is important method to plant disease control because it save

cost and it is more safety than using chemical. Biological control of plant diseases

can occur via several distinct mechanisms, including competition for nutrients

between a pathogen and harmless species, parasitism, and production of antibiotics.

Mechanisms leading to biological control of plant pathogens are complex and may

occur by many routes. Plant pathogens may be suppressed by events that reduce the

potential inoculum level of the pathogen in the environment, or by competitive or

parasitic interactions among organisms, or by competition for limiting resources.

Competition may occur at any point in the infection cycle, from its initiation outside

the host, through invasion and growth inside the plant. Some fungi such as species of

Trichoderma exhibit mycoparasitism, attacking and killing hyphae or other parts of

pathogenic fungi. Some bacteria, actinomycetes, and fungi produce chemicals

(antibiotics) that actively repress the growth of other species, including pathogens.

Some fungi repress the growth of pathogens by out competing them for key resources

such as minerals, nutrients, oxygen, or water, either at or away from the site of initial

infection (Van Driesche and Bellows, 1996).

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a. The host ; The host population in its wild state was always involved in

biological control by being a part of the biological balance that helped keep the

pathogen suppressed. If the host or crop plant is highly susceptible to the pathogen,

severe disease losses will occur unless the environment is highly unfavorable or

antagonists suppress the pathogens. The mechanism of resistance is known in only a

few instances. In some cases, the host may be resistant because it stimulates

antagonists to grow in its rhizosphere. Resistant varieties may be rendered susceptible

by products of decomposition of organic matter in soil.

b. The pathogen or parasite ; Pathogens and parasites are generally more

sensitive to unfavorable a biotic factors than are saprophytes. Most pathogens invade

the host early in the disease and, being internal, are generally protected from

antagonists. In addition, secondary organisms may, however, invade diseased tissue

and rot it.

c. Physical environment ; Control of a disease through the inhibitory effects of

the physical or chemical environment directly on the pathogen would not be a type of

biological control, because it is not then operating through another organism.

However, the environment may favor the host and causes it to maintain its resistance

to facultative types of microorganism

Conditions of the environment may be made unfavorable to the pathogen or

vector. Tillage practices that modify the environment so as to favor antagonists are

certainly part of biological control.

An admittedly oversimplified schematic diagram of the major factor-groups

acting to produce plant disease is shown in Figure 2.1. Each factor-group is

represented by a disk, and the amount of overlapping indicates the degree of

interaction. Environment is collectively used to include several factors that might be

operative; it could also refer to a single controlling factor.

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Figure 2.1 Schematic diagram of interactions in major factor-groups in plant disease.

A. Severe disease loss. Susceptible crop moderately well adapted to the

environment; pathogen well adapted; antagonists not well adapted and

ineffective. Exemplified by the Fusarium wilt diseases in acid sandy soils.

B. Slight disease loss. Susceptible crop well adapted to environment; pathogen

poorly adapted; antagonists moderately adapted and quite effective.

Exemplified by the Fusarium wilt diseases in alkaline clay soils.

C. No disease loss; biological control. Susceptible crop, antagonists, and

pathogen well adapted to environment. Antagonists have suppressed the

pathogen. Exemplified by Phytophthora cinnamomi root rot of avocado in

Queensland.

D. No desease loss; resistance. Resistant crop, antagonists, and pathogen well

adapted to environment. Host resistance prevents disease. Exemplified by

Fusarium wilts in any soil when the crop carries monogenic resistance (Baker

and Cook, 1974).

C

D B

A

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d. Antagonists ; Any disease control in which antagonists are involved is

biological control. Antagonism is now considered to include three types of activity:

* Antibiosis and lysis. Antibiosis is the inhibition of one organism by a

metabolic product of another. Although it is usually an inhibition of growth,

it may be lethal. The metabolite may penetrate a cell and inhibit its activity

by chemical toxicity. Lysis is a general term for the destruction,

disintegration, dissolution, or decomposition of biological materials.

Because of the variety of ways it can be produced, and the number of its

effects on plant cells, confusion has resulted in the literature.

∗ Competition. Competition was viewed by Clark (Baker and Snyder, 1965)

as “the endeavor of two or more organisms to gain the measure each wants

from the supply of a substrate, in the specific form and under the specific

conditions in which that substrate is presented when that supply is not

sufficient for both.” In essence, competition is for nutrients, particularly

high-energy carbohydrates, but also nitrogen, and possibly certain growth

factors.

∗ Parasitism and predation. Although the existence of this type of biological

control is not questioned, there is uncertainty about its actual importance

under field conditions. Fungi known to parasitize other organisms are

Rhizoctonia solani on Pythium, Trichoderma viride on Armillaria mellea,

several genera of trapping fungi on nematodes, Fusarium roseum on rusts.

The free-living nematode (Aphelenchus avenae), a ubiquitous fungivore,

thrusts its stylet into a hypha and injects digestive saliva that liquified the

contents, which are then sucked out through the stylet. Predatory nematodes

such as Seinura rapidly paralyze other nematodes by injecting saliva and

later sucking out the digested contents (Baker and Cook, 1974).

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Example of antagonistic microbes such as the activities of Trichoderma spp.

are inhibitory to a range of fungi. Trichoderma harzianum mycoparasitises the

mycelium of some other fungal species in soils, e.g. Rhizoctonia and Sclerotinia, and

inhibits the growth of others, e.g. Pythium and Fusarium. The fungus Gliocladium

virens mycoprasitises mycelium of Sclerotinia sclerotiorum (Isaac, 1992).

For plant pathogen, biological control is the reduction of disease by any of

these following action (Baker and Cook, 1974).

1. Reduction of inoculum of the pathogen through decreased survival

between crops.

2. Reduction of infection of the host by the pathogen.

3. Reduction of severity of attack by the pathogen.

2.1.2 Mechanism of fungal biological control

A number of the natural characteristics of the life-styles of fungi confer

qualities which make them potentially useful biological control agents against a

variety of pests and pathogens of plants.

There are several action of fungal biological control by following action.

(a) Competitive ability

The competitive activities of some fungi render them highly antagonistic and

ideal as potential combative organisms. In theory, at least, increased levels of such

species introduced to leaf surfaces would lower the potential rates of infection from

other pathogenic fungal species, which tend to be less competitive and aggressive.

(b) Antibiosis

Antibiosis is defined as the inhibition of the growth of a microbe by

substances produced and liberated by another microbe. The term most usually refers

to antibiotic activity. However, whilst it is relatively easy to prove that an organism

produces antibiotic in culture it is difficult to ascertain whether similar production

occurs under natural conditions, and even more difficult to establish a role for these

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compounds in competition within natural environments. Little antibiotic activity has

been detected in the soil environment and it has been suggested that these compounds

are degraded or adsorbed no to soil particles. Additionally, a strain newly isolated

from the environment may demonstrate antibiotic activity although this ability may be

rapidly lost on subsequent subculture.

(c) Mycoparasitism

Fungi which derive most or all their nutrients from another fungus are termed

mycoparasites. The term used to describe the direct parasitism of one parasite

(usually a primary parasite) by another is hyperparasitism. Fungal preparations are

now used and marketed commercially for control of insect pests and nematodes,

particularly in controlled, greenhouse conditions. All the major fungal taxonomic

groups contain mycoparasitic species. Biotrophic mycoparasites may have relatively

long-term associations with living cells of the invaded species; however, necrotrophic

mycoparasites often kill the target fungal-host cells prior to penetration and invasion.

Some mycoparasitic species are adapted to the exploitation of fungal spores,

either asexual or sexual resting spores. Exploitation of this ability, particularly the

mycoparasitism of sclerotia, would be of great agricultural and horticultural interest

since these structures are extremely long-lived and very difficult to eradicate from soil

(Isaac, 1992).

2.2 Endophytes

Endophytes was defined by Petrini (1991) as “all organisms inhabiting plant

organs that at some time in their life, can colonise internal plant tissues without

causing apparent harm to the host”. This definition therefore includes symptom less

latent pathogens and those fungi which also hame an epiphytic phase of their life

cycle. Wilson (1995) provided a “working definition” of the term by analyzing the

different levels of endophyte association and stated that “endophytes are fungi or

bacteria which, for all or part of their life cycle, invade the tissues of living plants and

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cause unapparent and asymptomatic infections entirely within plant tissues but cause

no symptoms of disease”. Endophytic fungi are as highly specialized on their hosts as

pathogenic fungi, but in contrast they colonize internal host tissue without manifest

symptoms (Petrini and Ouellette, 1994).

Fungal endophytes provide the protection to their host. For examplae, fungi in

redwood may function as antagonists or stimulators to pathogens (Espinosa-García et.

al., 1996). However, colonization or infection by endophytic organisms cannot be

considered as causing disease, because a plant disease in an interaction between the

host, parasite, vector and environment and symptoms are a result from the interaction

(Rossman, 1997). The study of endophytes of tropical plants has received much

attention because endophytes are believed to be both diverse and to provide an

excellent potential source of biologically active novel compounds (Dreyfuss and

Petrini, 1984; Hyde, 2001)

2.2.1 Characteristics of endophytes

Endophyte produced alkaloids and other mycotoxins appear to be responsible

for the resistance of plants. Several reviews discuss secondary metabolite production

by endophytic fungi in graminicolous and non-graminicolous hosts (Miller, 1986;

Clay, 1988, 1991; Petrini et. al., 1992). Furthermore, endophytic fungi alter

relationships between diversity and ecosystem properties as shown in Figure 2.2 each

species is represented by a circle that shows the amount of resources it exploits along

two resource axes. Grey circles represent the grass species. If the grass hosts an

endophyte, it experiences an increase in resource acquisition relative to its uninfected

state. This increase will be the strongest when resources are most limiting (i.e. at

greater levels of species diversity). As the infected grass is superior to the uninfected

grass in resource acquisition, it can more strongly reduce the amount of resources

available to other species (Jennifer et. al., 2004).

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Endophytes, in culture, can produce biologically active compounds (Brunner

and Petrini, 1992) including several alkaloids, paxilline, lolitrems and tetraenone

steroids (Dahlman et. al., 1991). When Eupenicillium spp. was isolated from healthy

leaves of Murraya paniculata (Rutaceae) after surface sterilization, the fungus was

cultivated in sterilized white-corn, where the spiroquinazoline alkaloids

alanditrypinone, alantryphenone, alantrypinene and alantryleunone were produced

(Barros, 2005). Endophytes, 155 fungi (91.7%) and 52 bacteria (64%), were found to

produce xylanase. The inside part of plants is a novel and good source for isolating

xylanase producers in comparison with soil (Suto et. al., 2002). Ergopeptine,

peramine, and pyrollizadine based loline alkaloids are produced from Acremonium

spp, these alkaloids are biologically active against numerous species of insects,

including aphids (Johnson et. al., 1985; Prestidge et. al., 1982; Siegel and Schardl,

1991). The alkaloids not only serve as feeding deterrents, but also decrease the

reproduction and growth of the insects. Antibiotic compounds have also been

produced in culture by endophytes (Fisher et. al., 1984a, b) and plant growth

promoting factors have been recovered (Petrini et. al., 1992).

Furthermore, endophyte was produced hormone for growth of plant. For

example, root growth and morphology are affected by some auxin-producing

mycorrhizae and Rhizobium bacteria (Leopole and Kriedemann, 1975). Auxin is a key

hormone in regulating plant growth, differentiation, and apical dominance and is

produced mainly in shoot meristems and expanding leaves. The endophyte is most

prevalent near these organs. The plants from which the endophytes were isolated

produced 24% greater biomass than did their nonsymbiotic isolines. Auxin

concentrations in both plant types were unaffected by endophyte infection. May be

expressions of differing defects on auxin concentrations or antagonisms by other

hormones such as gibberellic acid (Bacon and White, 1994). Endophytic

microorganisms exist within the living tissues of most plant species. They are most

abundant in rainforest plants. Novel endophytes usually have associated with them

novel secondary natural products and/or processes. For example, muscodor is a novel

endophytic fungal genus that produces bioactive volatile organic compounds (VOCs).

(Demain and Dijkhuizen, 2006)

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Evidence for the presence of endophytic fungi in many plants has been

provided solely on the basis of isolations of the fungi from surface-sterilized tissue.

From ecolofical and floristic analyses of a broad range of host species, it is apparent

that endophytic fungi comprise a unique and complex ecological group distinct from

obligate parasites and saprobes, although the ecological role of many endophytes is

not well understood (Petrini, 1994).

Figure 2.2 Graphical model depicting how mutualistic endophyte symbiosis in a

common grass species can alter the relationship between species diversity and

ecosystem functioning (Jennifer et. al., 2004).

2.2.2 Host specificity

The degree of host specificity which operates in the endophytic fungi is not

yet clear. Some species are commonly occurring and may be isolated from various

host plant species and from different locations with differing environmental

conditions. In general terms, the geographical occurrence of endophytes is related to

the distribution of host species. In some cases almost all individuals in a plant

population may be infected by endophytes. Cladosporium spp., Nodulisporium spp.

and Pleospora spp. are common. Some endophytes, however, do not show such a

wide species range and are often isolated from plants of the same family or closely

related families. Other species are only rarely detected (Isaac, 1992).

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The degree to which endophytes are tissue or organ-specific is also not yet

clear. Some species are most commonly isolated from similar tissues, particularly the

endophytes of conifer needles. In other cases the occurrence is less distinct.

Howerver, only limited surveys have been carried out to date (Isaac, 1992).

2.2.3 Isolation of endophytes

The isolation and identification of endophytic species involves very careful

surface sterilistion of host plant tissues followed by incubation on a range of media to

encourage outgrowth of isolates and their subsequent sporulation, so that

identification can be carried out. It is often difficult to satisfactorily establish

endophytic status for the isolates which are obtained. It is not easy to ensure the

exclusion of spores or hyphal fragments which may escape the sterilization (Petrini,

1986). Some epiphytic species may penetrate host tissues and additionally some

endophytic species sporulate in culture, although many isolates grow very slowly and

a considerable incubation time (sometimes many months) is often required. Fungal

endophytes of conifers are common although many belong to little known species,

probably because these fungi are very inconspicuous and are rarely collected. Some

species live almost entirely within a host plant cell. Rate of endphyte infection

increase with ageing of plants and plant populations. It is also possible that some

plant species may not occur naturally without endophytic fungal infections (Isaac,

1992).

.

Techa (2001) studied fungal endophytes associated with the palms

investigated at two sites within Doi Suthep-Pui National park, from different tissue

types (petiole, leaf, lamina and leaf veins). The endophytic fungi isolated included

Xylariaceous taxa (20 morphotypes), sterile mycelia (11 morphotypes), eight

unidentified hyphomycetes and twelve identified taxa. Likhittragulrung (2003)

studied endophytic fungi were isolated from healthy leaves, braches and petioles of

tomato, chili and devil’s fig plant, collected in Muang, Saraphi, Mae Rim and Doi

Saket districts, Chiang Mai province, Muang district, Lamphun province and Thoeng

district, Chiang Rai Province. All 611 endophytic fungi were recovered and grouped

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in to 20 taxa. Jansa and Vostka (2000) isolate of more than 200 strains of endophytic

fungi from the roots of several host plants belonging to order Ericales (Vaccinium,

Calluna, Rhododendon, Empetrum, etc.), followed by a successful attempt to verify

ericoid mycorhiza status of some of these fungal isolates under aseptic conditions. A

total of 131 endophytic actinomycete strains were successfully isolated from surface-

sterilized banana roots. These isolates belonged to Streptomyces, Streptoverticillium

and Streptosporangium spp. The remaining 2 isolates were not identified. About

18.3% of the isolates inhibited the growth of pathogenic Fusarium oxysporum f. sp.

cubense on banana tissue extract medium (Cao et. al., 2005). A total of 150

endophytic fungi were isolated from stems of cacao. The fungal community was

identified by morphological traits and rDNA sequencing as belonging to the genera

Acremonium, Blastomyces, Botryosphaeria, Cladosporium, Colletotrichum,

Cordyceps, Diaporthe, Fusarium, Geotrichum, Gibberella, Gliocladium,

Lasiodiplodia, Monilochoetes, Nectria, pestalotiopsis, Phomopsis, Pleurotus,

Pserdofusarium, Rhizopycnis, Syncephalastrum, Trichoderma, Verticillium and

Xylaria (Rubini et. al., 2005). Ganley and Newcombe (2006) investigated the

transmission of diverse fungal endophytes in seed and needles of Pinus monticola,

western white pine. They isolated 2003 fungal endophytes from 750 surface-

sterilized needles. In contrast, only 16 endophytic isolates were obtained from 800

surface-sterilized seeds. Twenty isolates of the endophytic actinomycetes were

isolated from the leaves and stems of healthy jujube plants in Chiang Mai Province by

using IMA-2 medium (Divarangkoon, 2004). Wathaneeyawech (2004) isolation 283

isolates of endophytic fungi from the corn leaves using triple surface sterilization

technique. Five hundred and fifty seven of endophytic fungi of 90 taxa were obtained

from nutgrass, cogon grass and common reed (Jailae, 2003).

2.2.4 Biological control of endophytic fungi

Endophytic fungi can be used to control fungal pathogenic in plant for the

safety of the environment and human. Many research work studies ability of

endophyte for used control pathogenic in plant. For example, Streptomyces sp. strain

S96 isolated from surface-sterilized banana roots inhibited the growth of pathogenic

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Fusarium oxysporum f. sp. on wilt pathogen in banana (Cao et. al., 2005). Samuels et

al. (2006) isolated Thichoderma theobromicola and T. paucisporum from cocao to

control Moniliphthora roreri causing frosty pod rot disease in cacao. Dhingra et al.

(2006) selected an endophytic non-pathogenic isolate of Fusarium oxysporum (NPFo)

and an antibiotic producing rhizosphere/rhizoplane (RS-RP) competent fluorescent

pseudomonas to suppress Fusarium yellow (F. oxysporum (Schlecht) f. sp. phaseoli

Kendrick and Snyder ) of bean (Phaseolus vulgaris L.). Rubini et. al. (2005) studied

endophytic fungi that can be used to inhibit Crinipellis perniciosa causes of Witches’

Broom Disease of Cacao which is the main factor limiting cacao production in the

Americas. They found that Gliocladium catenulatum reduced the incidence of

Witches’ Broom Disease in cacao seedlings to 70%. Park et. al. (2005) studied the

Chaetomium globosum strain F0142, which was isolated from barnyard grass. It was

found that the fungi showed potent disease control efficacy against rice blast

(Magnaporthe grisea) and wheat leaf rust (Puccinia recondita). Waller et. al. (2005)

studied potential of Piriformospora indica to induce resistance to fungal diseases and

tolerance to salt stress in the monocotyledonous plant barley. The beneficial effect on

the defense status is detected in distal leaves, demonstrating a systemic induction of

resistance by a root-endophytic fungus. The systemically altered “defense readiness”

is associated with an elevated antioxidative capacity due to an activation of the

glutathione-ascorbate cycle and results in an overall increase in grain yield.

Wathaneeyawech (2004) studied endophytic fungi isolated from corn leaves for

inhibition Northern leaf blight disease of corn.

2.3 Phytophthora

2.3.1 Phytophthora as plant pathogens

There are about 60 species in the genus Phytophthora, all of them are plant

pathogens. Phytophthora or the plant destroyer is one of the most destructive genera

of plant pathogens in temperate and tropical regions, causing annual damages of

billions of dollars (Drenth and Guest, 2004).

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Phytophthora diseases have been well studied in the temperate regions of the

world, ever since the potato late blight epidemic in Europe in 1845 - 1847 provided

the impetus for the development of plant pathology as a scientific discipline.

Throughout the wet tropics, agricultural production of a large range of crops is

seriously reduced due to the wide range of Phytophthora pathogens causing a large

number of different diseases (Drenth and Guest, 2004).

There are a number of host and pathogen factors which, together with features

of their interactions, make Phytophthora diseases so troublesome in the wet tropics.

One of the important factors to consider is that the genus Phytophthora does not

belong to the fungal kingdom. It is an Oomycete, closely related to diatoms, kelps and

golden brown algae in the Kingdom Stramenopila (Beakes, 1998). These organisms

thrive in the environments found commonly in the wet tropics.

Classification of Phytophthora was described by Kirk et. al. (2001) as

followed.

Phylum Oomycota

Class Oomycocetes

Order Peronosporales

Family Pythiaceae

Genus Phytophthora

Mycelium is generally coenocytic with no or a few septa, in host often with

haustoria. Hyphae 3 – 8 µm up to 12 µm, irregularly swollen undulate or gnarled;

sometimes with characteristic swellings; initial branching at right angles to parent

hypha and often swollen for a short distance. Chlamydospores usually spherical,

intercalary, sometimes terminal, wall smooth, up to 2 µm thick, hyaline at first.

Sporangiophores usually undifferentiated apart from a few spp. Where branching is

reminiscent of Peromospora but with nodal swellings; branching sympodial or

irregular and from below the sporangium or from within an empty one. Sporangia

usually terminal, single on long hyphae in sympodia or within (or just beyond) an

evacuated sporangium; ellipsoid, ovoid, obpyriform or limoniform (when shed); apex

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differentiated by an internal hyaline thickening of the inner wall of a depth (up to 6

µm) constant for the sp. and sometimes protruding to form a papilla; wall smooth up

to 2 µm thick; non-caduceus or shed with a pedicel. Germination by zoospores

emerging individually through apex (free at once or held momentarily in an

evanescent vesicle) or by a germ tube. Zoospores hyaline, ovoid to phaseoliform,

biflagellate, anteriorly directed tinsel (shorter) and posteriorly directed whiplash

(longer); when motility ceases the spherical cyst may show repetitional emergence but

not diplanetism. Oogonium usually terminal, spherical or tapering to the stalk,

delimited by a thick septum; wall hyaline, thin, becoming thicker and often yellow to

brown, mostly smooth occasionally tuberculate or reticulate. Antheridium usually

single, monoclinous or diclinous, spherical oval, cravat or short cylindrical (Figure

2.3), often angula; amphigynous or paragynous (sometimes both), if latter usually

applied to the oogonium close to the stalk. Oospore single more or less filling

oogonium, spherical, smooth, hyaline (sometimes faintly yellow), outer wall very

thin, inner wall 0.5 – 6 µm thick, when mature with large central globule

(Waterhouse, 1963; Holliday, 1980). Phytophthora can produce both asexual and

sexual spores. Asexual sporangia emergedirectly from the hyphae through structures

known as sporangiophores. Under optimal conditions of temperature and moisture,

sporangia release swimming spores. Sexual spores, or oospores, are formed when the

male structure, antheridium, associates with the female, egg bearing oogonium. Some

species of Phytophthora are self-fertile or homothallic, whereas others are self-sterile

or heterothallic. Heterothallic species are divided into A1 and A2 mating types and

crossing occurs when these two type of strains contact each other (Kronstad, 2000).

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Figure 2.3 Phytophthora sp. A. Sporangiophores penetrating a stoma of a potato

leaf. B. Sporangial contents dividing an releasing zoospore. C. Intercellular mycelium

from a potato tuber showing the finger-like haustoria penetrating the cell walls.

(Webster, 1980)

All Phytophthora species need high humidity for sporulation and the

germination of sporangiospores and zoospores to initiate infections. Frequent or

seasonal heavy rainfall, and high levels of humidity, are common throughout the

tropical lowlands. Tropical highlands have the added problem of heavy mist and dew

during the morning and/or late afternoon, producing free water throughout the night

and providing almost daily opportunities for sporangiospores to be formed,

transported and start new infections.

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Another important factor in the pathogenicity of Phytophthora is that

sporangia release motile zoospores that are attracted by chemotaxis and electrotaxis to

the roots of their host plants. The ability to seek out susceptible host tissue, coupled

with zoospore motility, makes these propagules extremely efficient, even at low

numbers (Drenth and Guest, 2004).

Factors involved Phytophthora to be formidable plant pathogens are:

a. The ability to produce different types of spores such as sporangia and

zoospores for short-term survival and spread, and chlamydospores and

oospores for longer term survival.

b. Rapid sporulation on host tissue within 3 - 5 days of infection. This results

in a rapid build-up of secondary inoculum in a multicyclic fashion, leading

to epidemics under suitable favorable environmental conditions.

c. Ability of zoospores of Phytophthora to be attracted to root tips through a

chemical stimulus (positive chemotaxis) as well as root-generated electric

fields (electrotaxis), coupled with the mobility of zoospores to actually

swim to the actively growing root tips, encyst, and infect young,

susceptible root tissue.

d. Ability to survive in or outside the host tissue as oospores are also known

to survive passage through the digestive systems of animals such as snails.

e. Production of sporangia, which can be airborne and may travel reasonable

distances in raindrops, run-off and irrigation water, and on wind currents,

to infect neighboring fields. These sporangia can directly infect host tissue.

These same sporangiospores also have the ability to differentiate into 4 -

32 zoospores under humid and cool conditions and cause multiple

infections from the one sporangium. Nevertheless, zoospores can travel

only short distances, as they are susceptible to desiccation.

f. Phytophthora pathogens belong to the Kingdom Stramenopiles

(Hawksworth et. al., 1995) and as such have different biochemical

pathways to the true fungi. Many fungicides are therefore not very

effective against Phytophthora pathogens.

21

g. Phytophthora pathogens thrive under humid and wet condition, which

makes them difficult to control, as protectant fungicides are difficult to

apply and least effective under such conditions.

2.3.2 Plant diseases causes by Phytophthora

Phytophthora spp. cause the most serious soilborne diseases of citrus. These

fungi are worldwide in distribution and cause citrus production losses in irrigated, arid

arese as well as in areas with high rainfall. Diseases caused by Phytophthora spp.

include damping-off in seedbeds and foot rot, gummosis, and root rot in nurseries and

orchards. Brown rot of fruit occurs in groves and continues to spread in

packinghouses, causing further losses (Timmer, et al. 2000).

Phytophthora pathogens can cause many different diseases and disease

symptoms on a wide range of plant species. There are many species of Phytophthora,

some of which have extremely wide host ranges and are particularly destructive and

are therefore important plant pathogens. Some of the economically important

Phytophthora pathogens and their major host plants are listed in Table 2.1

Table 2.1 Host plant and Phytophthora species (Kronstad, 2000; Drenth and Guest,

2004)

Phytophthora species Major host plant(s)

P. capsici

P. cinnamomi

P. citrophthora

P. fragariae var. fragariae

P. infestans

P. palmivora

P. sojae

P. botryosa

P. nicotianae

Pepper

Avocado

Citrus

Strawberry

Potato and tomato

Cocoa, papaya, coconut, black pepper, wild

durian, rubber, longan, mango, pineapple and

palm species

Soybean

Rubber

Citrus, durian, pineapple and black pepper

22

The disease symptoms most of often encountered are discussed as follow.

A. Late blight disease which cause blackening of leaves and other plant organs

and has often been applied particularly to infections which spread very rapidly

Probably the most famous of the blight diseases is that caused by Phytophtrora

infestans on potato, tomato and Solanaceae Late blight of potato caused widespread

and devastating famine in Ireland between 1845 - 1850. Phytphthora late blight kills

the foliage and stems of potential crop plants in the field (Isaac, 1992). And cracking

is a serious problem since molds and rots find ready entrance to reduce the quality of

the fruit and the picked product (Work and Carew, 1955). Late Blight – General and

serious in humid regions and in cool, wet seasons. Irregular, greenish-black, water-

soaked, rapidly enlarging, greasy spots on leaves, petioles, and stems. In moist

weather, a whitish-gray downy growth appears, mostly on underside of leaves

Infected foliage soon dries, turns brown, brown to black round lesions, with yellow-

green margins first appear at the tips or sides of the leaves and withers, Tomato fruit

spots are greasy and dark green to brown or pearly black, fruit are firm with a

corrugated appearance. The infection spreads, the fruits rot and become covered with

a whitish growth (Figure. 2.4) (Sburtleff, 1966; Centre for Overseas Pest Research,

1983).

Figure 2.4 Symptom of Late blight disease in tomato caused by P. infestans.

(www.mtvernon.wsu.edu/phth-team/diseasegallery.htm) [2006, October 16].

23

B. Brown rot disease caused from P. citrophthora on citrus. Phytophtrora may

infect citrus when its population build-up is much more than that of other species.

Blossoms wilt, turn brown, and rot. Leaves on twig tips suddenly wither and turn

brown. Twigs may die back from sunken, brown, girdling cankers, the bark color

darkens and the internal tissues decay extending into the wood (Figure. 2.5). Soft,

brown, rotted areas was occurred in fruit. Affected areas may later be covered by

powdery tufts of gray to tan mold (Sburtleff, 1966; Mukhopadhyay, 2004).

Losses from brown rot result primarily by rotting of the fruit in the orchard,

although serious losses may also appear during transit and marketing of the fruit.

Yields may be reduced also by destruction of the flowers during the blossom blight

stage of the disease. Twig infections do not always cause losses directly, but may

cause indirect losses by furnishing inoculum for fruit infections. In severe infections,

and in the absence of good control measures, 50 - 70% of the fruit may rot in the

orchard and the remainder may become infected before it reaches the market (Agrios,

1972). If such fruit are packed, they may cause other fruit in the same container to

become infected (Timmer et. al., 2000).

Figure 2.5 Symptom of Brown Rot disease in citrus caused by P. Citrophthora.

(www.giswebr06.ldd.go.th/knowledge/agrilib/plant/tangerine/distangl.htm)

[2006, October 10].

24

C. Black Stripe disease in rubber caused from Phytophtrora. Several species

of Phytophthora have been reported to be responsible for black stripe. The common

species are: Phytophthora palmivora (Butl.) Butl., P. meadii Mc Rae, P. botryosa

Chee (Drenth and Guest, 2004).

The early symptoms of black stripe are not obvious: a series of sunken and

slightly discolored areas just above the cut. Later, vertical fissures appear in the

renewing bark; when these are removed, dark vertical lines are visible. As the

infection progresses, the stripes coalesce forming broad lesions, finally spreading the

full width of the panel. When the disease is severe, it extends vertically in the wood

as far as 15 cm below the tapping cut and 2-5 cm upwards on the regenerating bark.

Pads of coagulated latex sometimes form beneath the bark causing extensive bark

splitting and bleeding (Figure 2.6).

Occasionally, infection occurs on untapped bark resulting in a wound, called

"canker". This may arise on bark previously affected by black stripe or on wounds

caused by spouts or wires. The early symptoms of canker are not obvious, but, in the

more advanced stage, the bark bursts and latex oozes out. Pads of coagulated latex

form under the bark causing it to bulge and split open.

Figure 2.6 Symptom of Black Stripe in rubber caused by Phytophtrora palmivora.

(www.giswebr06.ldd.go.th/knowledge/agrilib/plant/black.html)

[2006, October 10]

25

D. Root rot disease, seedlings of many plants is very susceptible to root rot

and damping off caused by Phytophthora. The early symptoms are the wilting and

yellowing of young seddlings. General symptoms of root rot are that plants appear

water stressed, chlorotic, and are often stunted in their growth. New leaves are often

small and show a light green to yellow colour and wilting occurs even in the presence

of sufficient water. Affected root tissue is soft, water soaked and discoloured to dark

brown rather than the creamy white colour of healthy roots. Advanced root rot leads

to the lack of secondary and tertiary roots and a lack of healthy root tips (Drenth and

Guest, 2004).

2.3.3 Phytophthora infestans

Phytophthora infestans, which is worldwide, causes the classical and

extensively investigated late blight of Irish potato and tomato (Holliday, 1980).

Sporangiophores differentiated from the mycelium (in the host) by being broader and

having a small swelling at the point of formation of each sporangium. Sporangia

abundant on the host and on solid media, ellipsoid, ovoid or (when shed) limoniform,

with a tendency to taper to the base, 19x29 (max. 31x59)µ, deciduous, pedicel short;

papilla not very protuberant, apical thickening less than hemispherical, usually 3-3.5µ

(Holliday, 1980) (Figure 2.7). P. infestans is a heterothallic fungus, that is, sexual

reproduction takes place by means of antheridia and oogonia of opposite mating

types. There is also evidence for hormonal of chemical control of sexual reproduction

in heterothallic species. In P. infestans, the oogonia penetrate and grow through the

antheridium developing into a globose structure above the antheridium. This type of

development is known as amphigynous development. Both the oogonia and

antheridia are multinucleate in the beginning but as they mature, single nucleus is left

that probably undergoes meiosis before fertilization. Migration of a single antheridial

nucleus to the oogonium occurs through the oogonial wall but fusion between the two

is delayed until the oospore wall is mature. After a rest period of several weeks, the

oospore germinates by means of a germ tube that usually terminates in a germ

sporangium. Zoospores produced in the sporangium give rise to new thalli (Johri,

2005) (Figure 2.8). Below 15๐C uninucleate zoospores are produced, whilst above

26

20๐C multinucleate germ tubes arise. With increasing age, sporangia lose their

capacity to produce zoospores. Direct germination is preceded by resorption of the

flagella, formed inside the sporangia (Webster, 1980).

Figure 2.7 Sporangium and sporangiophores of P. infestans, (a) sporangium and

(b) sporangiophores

a

b

27

Figure 2.8 Life cycle of Phytophthora infestans. Reproduced from Drenth (1994).

2.4 Production and biological control of tomato, citrus and rubber

Plant diseases are important because they cause economic losses to growers,

result in increased prices of products to consumers, and they destroy the beauty of the

environment (Agrios, 1972).

2.4.1 Tomato

The tomato belongs to the family Solanaceae (also known as the nightshade

family), genus Lycopersicon. The Solanaceae family includes other important

vegetable crops such as chilli and bell peppers, potato and tobacco. The Lycopersicon

genus includes a relatively small collection of species: the cultivated tomato L.

esculentum Mill. and several closely related wild Lycopersicon species, namely L.

esculentum var. cerasiforme, L. pimpinellifolium (Jusl.), L. cheesmannii, L.

parviflorum, L. chmielewski, L. hirsutum Humb., L. chilense Dun. and L. peruvianum

(L.) Mill. (Taylor, 1986).

28

Tomatoes are one of the most important horticultural crops both temperate

and tropical regions of the world, widely produced and consumed ‘vegetables’ in the

world, both for the fresh fruit market and the processed food industries. Furthermore,

tomato fruits or plants are occasionally used for decoration or ornamental value

(Heuvelink, 2005). It is easy to grow and nearly every home garden has it. It is most

gratifying to the palate, fresh or cooked; soft and grainy, smooth and juicy in texture.

In addition to the condiments, puree and paste are manufactured in commercial

quantities. A large share of the processed tomato pack is now sold as juice but

preservation by freezing has not been successful (Work, 1952). Their popularity

stems from the fact that they can be eaten fresh or in a multiple of processed forms.

Three major processed products are: (1) tomato preserves (e.g. whole peeled

tomatoes, tomato juice, tomato pulp, tomato purée, tomtopaste, pickled tomatoes); (2)

dried tomatoes (tomato powder, tomato flakes, dried tomato fruits); and (3) tomato-

based foods (e.g. tomato soup, tomato sauces, chillisauce, ketchup) (Heuvelink,

2005).

Tomatoes are commonly used as a ‘model crop’ for diverse physiological,

cellular, biochemical, molecular and genetic studies because they are easily grown,

have a short life cycle and are easy to manipulate (kinet and Peet, 1997).

The global production of tomatoes (fresh and processed) has increased by

about 30% in the last four decades. The annual worldwide production of tomatoes in

2003 has been estimated at 110 million with a total production area of about 4.2

million ha. In the future, global production is expected to increase for both fresh-

market and processing tomatoes. Based on investments made in the processing sector

and improvements in production systems and cultivars. China may be the main

source of such increases. Expansions in relatively inexpensive production areas,

together with in creasing production costs in more industrialized countries, are a

concern to many growers, especially those producing processing tomatoes

(Heuvelink, 2005).

29

Plant disease becomes the limiting factor in tomato production in many parts

of the world when cultivars with resistance to numerous diseases are not planted.

There are over 100,000 described species of fungi, and 20,000 of these are pathogenic

to plants or animals. Diseases caused by fungi such as alternaria stem canker caused

by Alternaria alternata, anthracnose may be caused by several species of the genus

Collectotrichum, black root rot by Thielaviopsis basicola, late blight caused by

Phytophthora infestans (Jones et. al., 1993), wilt caused by Fusarium oxysporum and

Verticillium dahliae, buckeye rot, stem rot, leaf blight caused by Phytophthora spp.

and leaf spot caused by Septoria lycopersici (Centre for overseas pest research, 1983).

Protection involves the use of cultural practices, manipulation of greenhouse

environments and planting time, regulation of soil moisture, adjustment of soil

reaction and fertility, control of insect vectors, and the use of protective chemicals

(Jones et. al., 1993). Cultural control measures of disease caused by Phytophthora

infestans include: (1) eliminating cull piles in the vicinity of tomato planting; (2)

destroying volunteer tomato plants; (3) using transplants that have passed a

certification programme and (4) applying fungicides when weather conditions favour

disease development (Heuvelink, 2005).

Example application of biological control for control disease in tomato using

Rahnella aquatilis control bacterial spot of tomato caused by Xanthomonas

campestris pv. This indicates that R. aquatilis reduced the deleterious effect and the

stress exerted by X. c. pv. vesicatoria on tomato seedlings. Foliar application of R.

aquatilis was the most effective method in disease reduction which could be attributed

to the direct effect of the antagonistic bacteria on the pathogen. The highest amounts

of fresh and dry weight ere obtained from seed treatment, which might suggest that

bacterial seed inoculation provides earlier protection than could be achieved with

foliar, soil or root treatment (El-Hendawy et. al., 2005). Control of tomato late blight

(LB) in Brazil is heavily based on chemicals. However, reduction in fungicide usage

is required in both conventional and organic production systems. Assuming that

biological control is an alternative for LB management, 208 epiphytic

microorganisms and 23 rhizobacteria (RB) were isolated from conventional and

30

organically grown tomato plants and tested for antagonistic activity against

Phytophthora infestans. Based on in vitro inhibition of sporangia germination and

detached leaXet bioassays, four EP microorganisms (Aspergillus sp., Cellulomonas

xavigena, Candida sp., and Cryptococcus sp.) were selected. These microorganisms

were applied either singly or combined on tomato plants treated or not with the RB

Bacillus cereus. On control plants, LB progress rate (r), area under disease progress

curve, and final disease severity were high. Lowest values of final severity were

recorded on plants colonized by B. cereus and treated with C. xavigena, Candida sp.

and Cryptococcus sp. There was no reduction on disease severity in plants treated

only with RB. Biological control of LB resulted in low values of r final severity.

Integration of biological control with fungicides, cultural practices, and other

measures can contribute to manage LB on tomato production systems (Júnior et. al.,

2006). The nonpathogenic Fusarium oxysporum strain Fo47 is an effective biocontrol

agent against Fusarium wilt of tomato caused by F. oxysporum f. sp. lycopersici

(Fuchs et. al., 1999).

To determine whether bacteria isolated from within plant tissue can have plant

growth-promotion potential and provide biological control against soil borne diseases,

seeds and young plants of oilseed rape (Brassicanapus L. cv. Casino) and tomato

(Lycopersicon lycopersicum L. cv. Dansk export) were inoculated with individual

bacterial isolates or mixtures of bacteria that originated from symptom less oilseed

rape, wild and cultivated. They were isolated after surface sterilization of living roots

and stems. The effects of these isolates on plant growth and soil borne diseases for

oilseed rape and tomato were evaluated in greenhouse experiments. We found

isolates that not only significantly improved seed germination, seedling length, and

plant growth of oilseed rape and tomato but also, when used for seed treatment,

significantly reduced disease symptoms caused by their vascular wilt pathogens

Verticillium dahliae Kleb and Fusarium oxysporum f. sp. lycopersici (Sacc.) ( Nejad

and Johnson, 2000).

31

2.4.2 Citrus

The true citrus fruit trees belong to the family of Rutaceae (Spiegel-Roy and

Goldschmidt, 1996). Citrus fruits fall in to several groups: sweet orange, sour orange,

mandarins and their hybrids, pummelos, grape fruit, lemons, and limes (Timmer et.

al., 2000).

Citrus fruits originated in South East Asia, including South China, north-

eastern India and Burma (Spiegel-Roy and Goldschmidt, 1996). Annual world

production of all citrus fruits is currently about 85 million metrictons. In many

countries, the crop is consumed as fresh fruit, but in some countries a major part of

the crop is marketed as a lightly processed, pasteurized, concentrated juice, jams or

confectionaries. (Timmer et al., 2000). Citrus trees and shrubs occur naturally

throughout the region, and seletions are widely cultivated. However, little is known

about the domestication process but it most likely started a long time ago since citrus

already were taken from southeast Asia for growing in the Mediterranean during the

great Greek civilization (Drenth and Guest, 2004).

Citrus is second only to the grape (of which most is used for wine) in the area

planted and in the production of fruit trees. Citrus planting (FAO Statistics) amount

worldwide to over two million hectares with citrus production estimated in 1992/3 at

76075000 tons. Brazil is by far the largest producers of oranges (19.7%), followed by

the USA (13.4%), China, Spain, Mexico, Italy, India and Egypt (Spiegel-Roy and

Goldschmidt, 1996).

Recent trends of citrus production and demand include all-year-round supply,

the increasing importance of industrial products (mainly concentrated fruit juice),

demand for seedless fresh fruit with a substantial increase in easy peeling. Citrus has

many uses, besides fresh fruit and consumer-processed fresh juice. Some of the uses

are by products of the processing industry and its main product concentrated fruit

juice. Products include canned fruit segments (mainly grapefruit and Satsuma

segments), citrus-based drinks, pectin, citric acid, seed oil, peel oil, essential and

32

distilled, citrus alcohol, citrus wines and brandies, citrus jams, jellies, marmalades and

gel products (Spiegel-Roy and Goldschmidt, 1996).

Citrus is subject to numerous diseases, some of which occur only in certain

environments, while others, like Phytophthora, pose a serious problem in all citrus-

growing areas. The common diseases, including postharvest fungal diseases such as

black root rot caused by Thielaviopsis basicola, wilt caused by Fusarium oxysporum,

Phytophthora spp. cause the most serious soilborne diseases. They are of worldwide

distribution. Losses are heavy in murseies (damping-off), in the orchard (foot rot

gummosis) and on the fruits (brown rot) (Spiegel-Roy and Goldschmidt, 1996;

Timmer et. al., 2000).

Example application of biological control in citrus. Citrus were propagated in

planting mixes amended with formulations of commercial biocontrol agents. Root

colonization by selected biocontrol agents was evaluated for citrus and found to be

generally between 76 to 100% in both greenhouse ebb and flow, and bench-produced

plants. Trichoderma harzianum and Bacillus subtilis being the most effective

uniformly among four tests. Four biocontrols reduced Phytophthora root rot on

citrus, and two biocontrol agents in combination reduced celery root rot caused by

Pythium and Fusarium spp., however, none improved above-ground plant growth or

health of citrus (Nemec et. al.,1996).

2.4.3 Rubber

Rubber belongs to the genus Hevea, species Hevea brasiliensis. The Hevea

brasiliensis is by far the most important of the species of Hevea. Ninety-nine per cent

of all the natural rubber produced in the world comes from this one species. Rubber

production in Hevea is entirely from the bark. The roots, wood, leaves, and other

portions of the plant do not enter into rubber production directly (Loreng, 1962).

33

Hevea brasiliensis or natural rubber was first introduced into Thailand from

Malaysia through Trang province, South Thailand, in 1900 or nearly a century ago. It

later spread to Chantaburi province, Southeast Thailand, in 1928. Because price of

rubber is fairly high compared to that of other crops, rubber growing area has,

therefore, increased rapidly from time to time (Anothai and Wate, 1995). Thailand is

the world’s largest producer of natural rubber since 1991. In 1993, a total rubber

production of Thailand was about 157 million tons or about 29.3 percent of the world

rubber production. Rubber is also one of the major exports and dollar earners of

Thailand. It contributed 3.1 to 3.8 percent of the country’s total export earnings

during the period 1992-1994 (Bank of Thailand, 1994). The subsequent

establishment and development of the rubber plantation industry to its present

outstanding position in tropical agriculture have been continuously aided by research

investigations. Production expansion has been maintained to meet the ever increasing

world demand for natural rubber (Verhaar, 1979).

Before pneumatic tyres came to dominate the world market for rubber,

important uses had developed for the vulcanized product. The manufacture and use of

boots and shoes made of rubber outdistanced that of all other rubber products and

continued to do so into the twentieth century. By 1900, world consumption lad

increased to 52, 500 long tons a year and consisted essentially of five general classes

of products (Loreng, 1962). (1) Boots and shoes. (2) Mechanical rubber good. (3)

Waterproof clothing other than boots and shoes had assumed great importance in the

Mexican war through the manufacture of rubberized ponchos. (4) The manufacture

and sale of drug sundries had become an important part of the rubber manufacturing

industry. Outstanding products were syringes, hot-water bottles, bandages, air pillows

and cushions, and atomizers. (5) The manufacture of hard-rubber goods had also

become important.

The majority of the rubber export at present is ribbed smoked sheet (71%),

block rubber (16%) and concentrated latex (10%), the other types of rubber (air dried

sheet, crepe and skim rubber) being small (4%). In 1993, about 130-236 metric tons

of rubber was consumed to produce many types of products. The main rubber

34

products of Thailand are tires and tubes for motorcars and airplanes (39%), gloves

(15%), rubber bands (10%), elastic (8%), tires and tube for motorcycles and bicycles

(7%), canvas shoes and foam candle (7%) and others (Anothai and Wate, 1995).

A plant disease caused by fungi is problem in rubber and economic especially

diseases of the stem and crown. The aerial portions of the Hevea tree are attacked by

numerous pathogens. Mostly of minor importance but some of great significance. In

general, it is convenient to divide these afflictions into those of the stem, of branches,

of bark, and of leaves. One pathogen, Phytophthora palmivora, attacks all of the

acrial parts and causes leaf-fall, twig dieback, and bark canker. Besides other diseases

such as oidium mildew caused by Oidium heveae and other leaf diseases caused by

species of Helnimthosporium, Scoletotrichum and Phyllosticta cause minor leaf-injury

in the East, and species of Alternaria, Pellicularia and Catacauma in the Americas.

(Loreng, 1962).

The control of diseases is an important function of estate management. Many

diseases attack Hevea, but the majority of them are of minor importance as they are

restricted in spread, lead to little mortality, and require little in the way of control.

Several have seriously affected stands of Hevea in localized areas, and a few have

resulted in epidemics of serious proportions (Loreng, 1962). Example application of

biological control in rubber. The fungitoxic effect of scopoletin was verified in vitro

on Microcyclus ulei where 2 mM concentrations were sufficient to strongly inhibit

germ tube elongation and conidium germination. In situ, 24 h after inoculation,

conidium germination and number of infection sites were lower in the most resistant

clones. The fungitoxic effect of scopolentin was tested on two other leaf pathogens of

the rubber tree, Colletotrichum gloeosporioides and Corynespora cassiicola.

Concentrations double or more than those tested on M.ulei were required for

inhibition of germination and germ tube elongation (Garcia et. al., 1995).