CHAPTER 2 REVIEW OF LITERATURE

Woody crops extend to all temperate areas. Apple one of the most important crop, reported to

originate in the temperate regions of the Western Asia. Commercially cultivated in India, largely

confined to the north-western Himalayan States of J&K, Himachal Pradesh, and Uttarakhand,

whereas northeastern states comprising Sikkim, Arunanchal Pradesh, Meghalaya, Manipur,

Nagaland and Assam. But due to the low production all of these states have megashare of less

than one percent in the total apple production. Different phytopathological threats, especially due

to the soil-borne fungi are gaining relevance. Various disease have been reported affecting apple,

among them soil borne disease such as white root rot (Dermatophora necatrix), collar rot

(Phytophthora cactorum), seedblight (Selerotium rolfsii), crown gall (Agrobacterium

tumefaciens), and hairy root (Agrobacterium rhizogens) cause significant losses to orchadists

both in nurseries as well as in orchards (Gupta and Sharma, 1999). A few fungi cause root rot in

woody plants in temperate areas; one of these is Rosellinia necatrix (Freeman and Stezjnberg,

1992), a cosmopolitan fungus from which quantitative data on losses are mostly lacking.

However, some studies have estimated increasing losses caused by this fungus in apple trees

(Malus spp.) (Agarwala and Sharma, 1966), glasshouse grapevines (Vitis vinifera) and Japanese

pear trees (Pyrus pyrifolia) (Ten Hoopen and Krauss, 2006).

2.1 Rosellinia necatrix a white root rot

Root rot is a very serious soil-borne disease infecting temperate fruits especially apple. It is

caused by Rosellinia necatrix Berl. ex Prill. (Anam. Dematophora necatrix Hartig) (Bharat,

2004). Rosellinia species are recorded all over the world, and are common in both temperate and

tropical regions (Petrini, 1993). Many species occur as saprobes, some live endophytically and

occasionally turn into pathogens and only a few species are known to occur as root pathogens.

Among the most well-known root pathogens are R. necatrix Prill. and R. desmazieresii (Berk. et

Br.) Sacc. ( ¼ R. quercina Hart.), mostly known from temperate zones, and R. bunodes (Berk. et

Br.) Sacc., R. pepo Pat. and R. arcuata Petch, known only from the tropics. Root diseases caused

by Rosellinia spp. occur on a wide variety of commercially important crops, trees and

ornamentals. The fungus, which occurs worldwide, is very aggressive and can kill infected trees.

However, few Rosellinia spp. appear to be of real economic importance, although quantitative

data on losses caused by Rosellinia spp. are greatly lacking. Agarwala and Sharma (1966)

estimated losses caused by R. necatrix in apple (Malus spp.) in the state of Himachal Pradesh,

India, to be at least $272,000 in 1963. This conservative estimate converts to more then $1.6

million dollars present time (Conversion CPI, Williamson, 2002). Of the root pathogens, R.

necatrix is the most common and occurs in all five continents in temperate, subtropical and

tropical zones (Petrini, 1993).

2.2 Biology of Rosellinia necatrix

Taxonomy

In 1844 the genus Rosellinia was named by de Notaris, (subdivision Ascomycotina;

class Euascomycetes; subclass Pyrenomycetes; order Sphaeriales, syn. Xylariales; family:

Xylariaceae) was identified but its systematic position within the pyrenomycetes has not

always been clear, mainly because the stromatic character of its fructifications was

unrecognised by many authors. This genus belongs to the family Xylariaceae, which

includes more than one hundred species, among them economically important root

rot pathogens such as Rosellinia arcuata Petch, Rosellinia desmazieresii (Berk. Br.)

Sacc. and Rosellinia pepo Pat. (Petrini, 1993).

Rosellinia necatrix: About 130 years ago, a disease called white root rot caused by R.

necatrix attracted attention for the first time when it caused damage in vineyards in

Germany and France (mentioned by Schnetzler, 1877 and cited by Behdad, 1975).

The first scientific studies of the fungus, D. necatrix, previously known as

Rhizomorpha necatrix, were made in Germany (Hartig, 1883). The anamorph state

was described as a severe root pathogen in vineyards by Hartig (1883). Berlese

(1892) concluded that Dematophora necatrix was a Rosellinia anamorph; Prillieux

(1902) linked D.necatrix with R. necatrix and Hansen et al., (1937) showed this

to be true. Perez et al., (2003) also confirmed the relationship between D. necatrix and

R. necatrix. Prillieux (1904) was the first to give a detailed description of the

teleomorph together with its disease symptoms. A description of the teleomorph

development is given by Nakamura et al., (2000). Martin (1968) combined the species

epithet with Hypoxylon but mistakenly cited Hartig as the authority of the

basionym, that being the reason why R. necatrix Prill was long cited as R. necatrix

(Hart.) Berl (Thon et al., 2000).

Rosellinia spp. are differentiated on the basis of their teleomorphic structures.

Generally, Rosellinia spp. possess smooth perithecia with dark, one-celled

ascospores. Size and shape of ascospores and perithecia are useful parameters for

species identification. However, a problem in identifying Rosellinia species may arise

as it can be difficult to obtain the teleomorph. Therefore, identification is often

based on vegetative structures alone. The thallus of Rosellinia necatrix, on culture

media, t h e young mycelium of R. necatrix is initially white and cottony but with

age it becomes brown–black in colour. Pigmentation is located on cell walls and

depends on metabolic activity. The coloration is more intense as the malt

concentration in the medium is increased and less intense with increased light. In

general, R. necatrix is a fast growing fungus and its mycelium covers the surface of

the culture medium when it is incubated in the dark at 20–24°C. The most

important morphological characteristic of R. necatrix hyphae is the existence of

pearshaped or pyriform swellings immediately above the septum, which have

generally been used to identify the species a s t h i s i s t h e c h a r a c t e r i s t i c f e a t u r e

s h o w n b y t h e p a t h o g e n (Fig. 1). They have a diameter of up to 13 µ m (i.e., four-

or six-fold the hyphal diameter), and are more abundant and larger in superficial

mycelia than in submerged mycelia (Khan, 1959; Makambila, 1978).

These swellings are characteristic feature and differentiate the pathogen from other

Rosellinia spp. A new method has been developed to induce and quantify

microsclerotia in R. necatrix. When the fungus is grown on PDA and exposed

continuously for 30 days to red (600–700 nm), or blue (350–530 nm) or artificial

daylight (380–770 nm) irradiation, induction of microsclerotia in mycelium is

observed (Sztejnberg et al., 1980). H o w e v e r , t h e efforts to cause their

germination have been unsuccessful (Jabareen and Sztejnberg, 1986). Synnemata

with conidiophores bearing conidia of R. necatrix can be produced by incubating

naturally infected roots in a moist chamber at 25 ºC for 2–3 weeks (Freeman and

Sztejnberg, 1992).

Fig 2.1: Cultural and morphological characteristics of R. necatrix.

Microphotograph of typical pear–shaped swelling of fungal

mycelia (Perez-Jimenez et al., 2006).

2.3 Geographical distribution

Found in mostly all temperate regions but can also be found in (sub) alpine and (sub)

tropical regions. Of the root pathogens, R. necatrix is the most common and occurs in

all five continents in temperate, subtropical and tropical zones (Saccas, 1956;

Sivanesan and Holliday, 1972b; Behdad, 1975; Petrini, 1993). R. pepo and R. bunodes

are more restricted and R. pepo, supposedly, more so than R. bunodes (Waterston,

1941; Saccas, 1956; Sivanesan and Holliday, 1972a). Both occur in tropical areas

in Central and South America, West-Africa, the West Indies, and Asia. R. arcuata is

less known and occurs primarily in Africa and Asia (Saccas, 1956; Sivanesan and

Holliday, 1972c). R. dezmazieresii is currently restricted to Europe (Ofong et. al., 1991;

Petrini, 1993). R. radiciperda, a pathogen of apple, is only known from New Zealand

(Petrini, 2003). R. minor, a pathogen of, e.g. Picea abies and P. excelsa, is known

from North America and Europe (Pacific Forestry Centre, undated; Cech, 1990;

Petrini, 1993). It has also been found growing on basidiocarps of Polyporus spp.

(Petrini, 1993). R. herpotrichoides Hepting and Davidson, causing needle blight on

Douglas-fir (Pseudotsuga menziesii), Sitka spruce (Picea sitchensis) and Tsuga sp. is

found in parts of Canada and the USA (Smith, Jr., 1989; Petrini, 1993).

2.4 Ecology

According to Petrini (1993), moisture is a very important ecological factor required by all

Rosellinia species for mycelial growth and fructuation. The root diseases caused by Rosellinia

spp. are often associated with acid soils, rich in organic matter (Ofong et. al., 1991; Mendoza et

.al., 2003). Mendoza et. al., (2003) confirmed that organic matter and pH are important factors in

disease development and control. The phosphate level, however, did not affect an isolate

collected from cocoa, but increased plant vigour was recorded due to phosphate applications.

Gupta and Gupta (1992) mention that R. necatrix in apple orchards is particularly severe in soils

with a pH of 6.1–6.5.

Field observations on spatial distribution of diseased trees suggest that the white root rot

fungus expands vegetatively through the soil from diseased roots to healthy roots (Matsuo and

Sakurai; 1954, Khan, 1959; Itoi et al., 1964) (Fig 2.)

2.5 Host range and symptoms

R. necatrix attacks about 170 species in 63 genera (Sztejnberg and Madar, 1980). Besides R.

necatrix has a very wide host range and is very destructive to many fruit tree crops, including

tropical and subtropical species (Plieqo et al., 2007). A list of 437 reports of fungus–host

combinations with specific references is available at http://nt.ars-

grin.gov/fungaldatabases/index.cfm, many of which are of economic interest. These include

tropical (avocado, coffee, citrus and mango) and temperate (almond, apple, fig, kiwi, grape,

olive, pear, peach, persimmon, sweet cherry and tea) fruit trees, nut tree crops (chestnut,

pistachio and walnut), small fruits, such as the strawberry, narrow leaf (cedar, fir, pine, sequoia

and yew) and wide leaf (holly, oak, poplar and elm) forest trees, herbaceous (daffodil and

paeony) and woody (azalea, camellia and rose) ornamental plants and field crops (alfalfa and

potato). Recently, R. necatrix was not only included in the fungi listed as regulated plant pests by

the US Department of Agriculture Animal and Plant Health Inspection Service (Cline and Farr,

2006), but simultaneously, this fungus was also included in the list of diseases already present in

Australia (New South Wales Government Gazette, 1 September 2006, available at

www.nsw.gov.au); since then, it has come to be considered as an emergent threat to many crops,

such as cotton, nuts, apples and pears (Department of Primary Industries, Vic., Australia, 2008,

available at http://new.dpi.vic.gov.au/agriculture/pests-diseases-and-

weeds/plantdiseases/fruitdiseases/white-root-rot) (Pliego et al., 2012).

Fig. 2.2: Characteristic macroscopic symptoms caused by Rosellinia necatrix on

avocado. (A) Healthy 15-year-old avocado tree. (B) Advanced aerial

symptoms of R. necatrix root rot on a 15-year-old avocado tree: dry leaves

attached to the wilted tree, sparse foliage and dry branches are observed. (C)

Hyphal strands and cords of R. necatrix on the soil surface. (D) Hyphal

strands and cords of R. necatrix spreading from colonized to healthy avocado

roots in an avocado pot plant. (E) Rosellinia necatrix spreading on the wood in

a symptomatic 15-year-old avocado tree (Pliego et al., 2012).

Other field crops (beans and cotton) and some weeds have also been killed when subjected to

artificial inoculations of R. necatrix (Pliego et al., 2012). In humid temperate regions, several

Rosellinia spp. caused needle blight of conifers (Francis, 1986), while others live endophytically

(Petrini, 1993). R. necatrix infection of avocado trees, often referred to as white root rot, is

clearly influenced by multitrophic interactions taking place in the rhizosphere. Among the plants

reportedly resistant to certain Rosellinia are: wheat (Triticum sativum), barley (Hordeum

vulgare), sugarcane (Saccharum officinarum), Guinea grass (Panicum maximum), coconut

(Cocos nucifera), Tradescantia spp., and passionfruit (Passiflora edulis) (Sztejnberg and Madar,

1980).

Observation of infected young roots, which are covered by a white mycelium, has revealed the

formation of mycelial aggregates migrating along the root surface and penetrating between cells

belonging to the cortical parenchyma, natural openings or through root wounds (Mantell and

Wheeler, 1973; Labrouche, 1982). In addition, penetration of R. necatrix into apple roots has

been reported to occur in various phases, each involving different forms of hyphal aggregates

(Labrouhe, 1982). Characteristic symptoms of this disease are rotting of roots, yellowing and

falling of leaves, wilting and finally death of the tree.

The first indications of the disease are the aerial symptoms displayed by the leaves. At first, the

leaf margin incurves and this is followed by a change in colour, first to red then to yellow.

Premature defoliation is common. In diseased trees there is an absence of new shoot growth and

fruits stop developing and many become mummified. These disease symptoms are therefore

similar to those caused by a number of root infecting fungi especially species of Phytophthora. It

is however the development of a white mycelium fan which first rots the small roots and then

invades larger roots, characteristic of Rosellinia infection. The surfaces of the infected roots are

covered by white mycelia cords which also extends under the bark and into the surrounding soil.

The white mycelium may be visible at ground level at the root crown when conditions are

favourable. Diseased trees are easily uprooted following the destruction of the root system.

Pieces of infected root transferred to damp chambers rapidly develop sheets of microsclerotia

and synnemata (columns of conidiophiores) on the diseased tissue. Depending on the age of the

tree and the severity of the infection, trees either die within a single season or linger on for two

or three seasons. Herbaceous plants collapse and wither within a few weeks of presenting the

diseased symptoms (Thienhirun et al., 1997).

2.6 Life Cycle of Rosellinia necatrix

The asexual life cycle of R. necatrix occurs through two different spore types: chlamydospores

and conidiospores Fig 3. Chlamydospores can be found only under exceptional environmental

conditions and are rarely found under natural and artificial conditions (Pérez-Jiménez et al.,

2003). These spores are almost spherical and are 15 mm in diameter; they originate by

condensation of the pyriform swellings in the protoplasm and subsequent formation of a cell wall

(Makambila, 1976). Conidia originate at the ends of synnemata of conidiogenous cells, which are

produced from either sclerotia (possibly related to pathogen survival in soil) or brown mycelial

masses. The length of the synnemata varies between 0.5 and 1.5 mm. Conidia are solitary, one-

celled, hyaline, elliptical, 3–5 mm in length and 2.5–3 mm in width, and are borne both apically

and laterally to the conidiogenous cells (Petrini, 1993).

The sexual reproductive structures of R. necatrix ascospores, are formed inside the perithecium

and, when they reach maturity, are expelled into a mucilaginous mass from the pore of the

papilla located at the top of the perithecium. These structures have been found on infected apple,

loquat and avocado tree roots (Makambila, 1976; Lin and Duan, 1988; De Sousa and Whalley,

1991; Pérez-Jiménez et al., 2003). Their diameter ranges from 0.89 to 1.78 mm, and their height

from 1.39 to 1.58 mm. Fresh perithecia are soft and spherical, with a gelatinous aspect and a

honey colour. As they age, they contract and acquire a brown–black colour and a dry aspect as a

result of hyphal and cell melanization. The asci are projected towards the interior of the

perithecium (Jiménez et al., 2003; Pliego et al., 2012).

Fig. 2.3: Life cycle of Rosellinia necatrix, covering the sexual and asexual

aspects of the cycle. The asexual life cycle occurs via two different spore

types, chlamydospores (not considered because they are rarely found

under natural conditions) and conidiospores. Conidia originate at the

ends of synnemata of conidiogenous cells, which are produced from

either sclerotia or brown mycelial masses. The sexual life cycle is

mediated by ascospores, which can be easily found in infected tissues.

The sexual reproductive structures of R. necatrix are formed inside the

perithecium and, when they reach maturity, are expelled. As they age,

they contract and acquire a brown–black colour and a dry aspect as a

result of hyphal and cell melanization. The asci are projected towards

the interior of the perithecium. Perithecia formation takes a long time

under natural conditions and has never been achieved previously in a

Petri dish (Pliego et al.,2012).

2.7 Genetics and variability of the fungus

According to recent publications the existence of a somatic incompatibility system in R.

necatrix has been described through barrage formation in pairings among both mass

and single ascosporic fungal isolates from infected avocado trees (Persea americana

Mill.). This study (Jimnez et al., 2002) revealed the existence of high genetic

diversity in this R. necatrix population. In Japan, Aimi et al., (2002) using molecular

biological methods (telomere-linked restriction fragment length polymorphism)

estimated the number of chromosomes of field isolates and single ascospore isolates

as about seven. As the number of chromosomes between mass and ascosporic

isolates appeared the same, they concluded that normal heterokaryon formation

does not occur in R. necatrix. Moreover, these authors found that telomeric

fingerprinting patterns essentially correspond to Mycelial Compatibility Groups and

Anastomosis Groups. Additionally, the existence of diverse double-stranded RNA in

massal isolates of the fungus, but not in single ascospores isolates, has been

demonstrated (Arakawa et al., 2002). Kanematsu et al., (2004) associated these

segments with hypovirulence and revealed that they have a viral origin. Ikeda et.

al., (2004) examined the diversity and transmission of these double-stranded RNA in

a large population of R. necatrix (Jimenez et al., 2006).

These recent studies indicate the existence of complex recognition systems in this

species, which determine population variability established in a determinate

area and have important epidemiological consequences (Jimenez et al., 2006).

2.8 Conditions influencing growth of Rosellinia necatrix and its pathogenecity

Anselmi and Giocelli (1990) studied factors influencing the incidence of R. necatrix in poplars

and found that R. necatrix spreads readily on loose soil with a high sand content. According to

their report soil moisture content near field capacity encouraged mycelia spread from tree but

that dry conditions rendered the trees liable to attack. Anselmi and Cellerino (1986) and Anselmi

and Giocelli (1990) emphasized the need to remove any woody or organic material from the soil

which might have been in contact with Rosellinia if control of the disease is to be successful.

The mycelium of R. necatrix is very sensitive to heat. In vitro studies show that the

optimum temperature for its growth is 22–24°C and it does not grow below 5°C and

above 32°C (Abe and Kono 1953; Araki, 1967; Mantell and Wheeler, 1973; Anselmi

and Giorcelli, 1990; Perez-Jimenez, 1997). Regardless of the osmotic water potential

for fungal growth in vitro, Lin and Duan (1988) established the optimum between 0

and 10 bars with a complete inhibition below 54 bars. Several researchers have

studied the effect of soil water content on R. necatrix growth. Anselmi and

Giorcelli (1990) demonstrated that soil moisture is the most important factor

influencing the growth of the fungus. In a sandy-silt soil, growth was optimal at

field capacity (moisture content at 21%) and was insignificant at maximum water

capacity (45% moisture) in anaerobic conditions. In general, the optimum is reached

when the soil is between 100 and 70% of the field capacity and growth decreases if

the soil water content is reduced (Araki, 1967; Mantell and Wheeler, 1973).

Rosellinia necatrix grows well in vitro at pH 5–8, and can even develop at pH 4

or 9. Although some variability can be found, depending of the culture medium

and the isolate, authors are generally in agreement about these values. Under

natural conditions, attack occurs in soils between pH 6 and 8 (Abe and Kono,

1953; Araki, 1967; Makambila et al., 1976; Anselmi and Giorcelli, 1990; Perez-

Jimenez, 1997).

Light has a strong inhibitory effect on R. necatrix growth. Thus, cultures incubated

in the light have a high lineal growth inhibition compared with cultures incubated

in darkness, depending on the temperature, culture age and wavelength used. The

mycelium developed in the light is superficial and weak, with scarcely branched

strands, but mycelium developed in darkness forms a consistent plectenchyma

(Makambila et al., 1976; Anselmi and Giorcelli, 1990).

Poor aeration is unfavourable for the occurrence of the disease, and growth of R.

necatrix mycelium is greatly retarded when the O2 content of the air is less than 10%

(Araki, 1967). Thus, as indicated above, in soils under maximum water capacity and

in complete anaerobiosis growth is insignificant (Mantell and Wheeler, 1973).

Nutritional requirements, saprotrophic behaviour and survival Rosellinia necatrix

grows poorly on synthetic media but on natural plant extract media containing

peptone, growth is vigorous. Vitamins, biotin, thiamine and inositol, asparagine,

glucose and some mineral salts are essential for fungal growth (Abe and Kono,

1955). Araki (1967) showed that R. necatrix possesses very high levels of cellulolytic

enzymes but very low levels of pectic enzymes. R. necatrix can efficiently

metabolise existing sugars into the vegetative cell; D-glucose, D-fructose, maltose

and starch, but not sucrose. Likewise, the hemicelluloses degradation products (D-

arabinose, D-xylose, D-galactose, D-mannose and D-arabinose), especially mannose,

are good carbon sources for the development of the fungus. Of the cellulose

compounds that have been studied, it has been found that cellobiose is a good

carbon source whereas carboximethylcellulose is not. R. necatrix can also

metabolise some fruit pectins (Melo and Ferraz, 1990). Lignin can be degraded and

used as a C source by R. necatrix, though the ability for degradation is influenced

by glucose concentration in the media (Cortizo et al., 1982). These authors indicated

that a synergistic effect occurs for C utilization when glucose and lignin are both

present in the growth medium (Jimenez et al., 2006).

Under laboratory conditions, the fungus can survive for a long time on plant

material, and has remained viable on an apple branch for 8 years (Thomas et

al.,1953). The fungus requires fresh vegetable debris rich in cellulose, and could

survived in pear branches for a period of 18 months. However, decrease in cellulose

c o n t e n t u p to 50% of the original could lead to the death of the fungus (Araki,

1967). Studies carried out with sterilized and non-sterilized soils show that R.

necatrix has a reduced colonization ability in the presence of other soil

microorganisms (Mantell and Wheeler, 1973).

R. necatrix development is encouraged when vegetable debris is present in the soil

because organic matter, which decomposes slowly activates the saprotrophic and

parasitic activity of the fungus. However, green amendments, such as soybean,

bean or pea, restrict fungal development (Bhardwaj et al., 2000). Rosellinia

necatrix can be present in a range of different soils (clay, calcareous, granite or

sandy) but it grows slowly in clay soils, with low oxygen content and strong

humidity changes (Khan, 1959). Where infection occurs, the attack is more

harmful when plants are water stressed (Mantell and Wheeler, 1973). Anselmi

and Giorcelli (1990) reported the influence of different environmental factors on

R. necatrix growth. They reported that, under controlled conditions, the fungus

is particularly favoured by loose or crumbly soils, where mycelial growth can

reach 6 mm/day. This contrasts with compact soil structures where growth could

be less than 1 mm/day. It is also favoured by moisture levels near field capacity,

and temperatures of 22–25°C, while it is slowed by a low or excessive water content

and by temperatures lower than 10°C or higher than 28°C. Moreover, it is

strongly inhibited by exposure to light or to dry air (Jimenez et al., 2006).

2.9 Isolation, culturing and storage

R. necatrix can easily be isolated from the surfaces of infected roots which are covered with

strands of white mycelium or from under the bark, where a fine, continuous layer of the

mycelium is usually present. Using a trapping technique with avocado leaf discs (Sztejnberg et

al., 1983a) or twigs of Populus sieboldii (Ito and Nakamura, 1984), it was possible to isolate R.

necatrix from soil samples. The leaf disc method described by Freeman and Sztejnberg (1992)

was used by the authors for trapping a Rosellinia isolate presumely R. bunodes from diseased

cocoa roots. The researchers used cocoa leaf discs as well as avocado leaf discs but were unable

to isolate the pathogen. The leaf disc method could be used to assess relative levels of pathogen

population of R. necatrix, and within certain limits, the inoculum density, as this parameter and

percentage colonized leaf discs are linearly correlated (Freeman and Sztejnberg, 1992).

R. bunodes and R. pepo can be collected from roots of diseased cocoa and coffee plants.

However, direct transference to potato dextrose agar (PDA) plates will often not be effective due

to contamination with strong competitors and mycoparasites that inhibit pathogen growth

(Fernandez and Lopez, 1964; Mendoza, 2000).

Therefore Fernandez and Lopez (1964), used acidified PDA (PDAA, pH ¼ 3.5) for isolating R.

bunodes. Plates were inoculated with portions from infected roots of which the exterior was

disinfected using mercury dichloride. Isolation of R. pepo was obtained by direct transference of

the mycelium to PDA plates with Rose Bengal at a concentration of 67 mg/l. Mendoza (2000)

investigated several methods to obtain Rosellinia isolates from diseased roots of cocoa, coffee

and potato. He incubated infected roots in vermiculite or wrapped in Kraft paper, and also

attempted direct plating techniques on several media: PDA, PDAA, water agar, and Czapek-Dox

agar (CDA) with Rose Bengal. Kraft paper and vermiculite facilitated the isolation process of

Rosellinia isolates of cocoa, whereas the only success in isolating Rosellinia from coffee roots

was obtained by direct plating on PDAA. This was the only medium on which direct isolation

was successful. R. necatrix isolates are easily transferred and need few to no special

requirements for storage. Freeman and Sztejnberg (1992) reported that R. necatrix stays viable

for up to 10 years when stored on PDA slants at 4 ºC. Some of their isolates even remained

viable for up to 8 years when stored at fluctuating room temperatures (10–30 ºC). Aranzazu

(1996) found that R. pepo loses its pathogenicity over time when constantly subcultured. Ten

Hoopen et al., (2004) investigated several ways to store R. bunodes, R. necatrix and R. pepo

collected from cocoa and potato. Storage in a refrigerator at 5 ºC on different substrates proved

possible for up to two years for R. necatrix and R. pepo. An isolate of R. bunodes proved fairly

difficult to store for periods longer than 6 months. Best results were obtained when this isolate

was stored under oil. Storage in liquid nitrogen is an obvious solution, however, specifically

subtropical and tropical isolates may be sensitive to cold storage and storage media as is

suggested by Ten Hoopen et al., (2004).

2.10 Identification of Fungal Pathogenesis

A rational approach to increase our antifungal arsenal relies on the identification of

novel targets involved in various aspects of fungal biology. Although gene products

necessary for virulence are seen as candidate targets, no genuine virulence factor in

opportunistic fungal pathogens has yet been identified. Attractive alternative antifungal

targets are to be found among gene products that are essential for fungal growth both in

vivo and ex vivo (Firon et al., 2003).

To produce disease-free propagative material, methods for rapid identification and detection

of the pathogen are necessary. However, to our knowledge no specific methods for R. necatrix

detection are available. Isolation of the pathogen from infected roots is difficult due to the lack

of selective media and the occurrence of a large number of saprophytic micro-organisms.

The avocado leaf disk colonisation method (Sztejnberg et al., 1987) has been utilised to

isolate and assess inoculum level of R. necatrix in naturally and artificially infected soils,

but it is a laborious procedure and avocado leaves are not available everywhere. In addition,

expertise is needed to identify the fungus (Petrini, 1993).

The polymerase chain reaction (PCR) provides a reliable alternative for the identification

and detection of fungal pathogens. Internal transcribed spacer (ITS1 and ITS2) regions

within ribosomal gene clusters are widely utilised to design species-specific PCR primers

(White et al., 1990). However, the large-scale applications of conventional PCR for the

detection of micro-organisms are limited, due to post amplification procedures necessary

to detect amplified fragments (Schaad et al., 1999). Real-time PCR combines the sensitivity

of conventional PCR with the generation of a specific fluorescence signal only when the

probe forms a stable hybrid with the complementary internal sequence of the amplicon.

Real-time PCR is widely used in medicine for the diagnosis of viral and bacterial infections

(Machida et al., 2000; Reischl et al., 2000) and there are increasing reports about its

application in plant pathology (Ippolito et al., 2000; Weller et al., 2000; Bates et al., 2001;

Cullen et al., 2001; Schena et al., 2002;). Several attempts have been made to generate

amplification systems in which the amplicon detection is based on fluorescence resonance

energy transfer (FRET) such as Taq-Man (Lee et al., 1993) and Molecular Beacons (Tyagi

and Kramer, 1996). Scorpion-PCR (Whitecombe et al., 1999) uses a uni-molecular

approach and the probe target binding is kinetically favoured over duplex reannealing and

thermodynamically favoured over intrastrand secondary structures. These characteristics

make Scorpion-PCR rapid and sensitive (Thelwell et al., 2000; Finetti et al., 2000).

Scorpion-PCR has been used to detect antagonistic (Schena et al., 2002) or pathogenic

fungi (Ippolito et al., 2000) and viruses (Finetti et al., 2000).

2.11 Understanding pathogenesis

An understanding of the mechanism of bacterial and fungal pathogenesis is dependent on the

identification and characterisation of the microbial genes products that influence the progression

of infection. The complexity of the changing cellular and tissue environments encountered by

invading pathogens has made this task very difficult to approach by physiology or biochemical

experimentation. A much more productive approach has been to use genetics and molecular

biology. With the development over the past two decade variety of a molecular genetics

screening and selection methods, it is now possible to identify and determine the functions of

major virulence determinants of many pathogens. This is clearly an important milestone because

our understanding of pathogenicity has often lagged behind the knowledge of host response to

infection. A detailed understanding of the sequence of events in pathogenesis can also be

expected to result in a more informed approach to the development of the new antibacterial and

antifungal drugs (Cohen, 1996).

Table 2.1: Approaches to the identification of microbial virulence determinants (Hensel et

al.,1996).

Approach Advantages Disadvantages/limitations

Expression-based:

cDNA cloning,

differential display

Promoter fusion

technology

Gene Transfer

Gene Comparison

Mutation-based:

Directed ( gene

disruption)

Random

UV/Chemicals

Transposons/REMI

STM

Sensitive, broadly applicable,

identifies genes induced during

infection

Rapid cloning of genes , can use

infected host for positive

selection

No genetic system required, rapid

cloning possible

Can clarify role of suspected

vcirulence determinant:

Easy to perform, generally

applicable

Direct tagging aids gene cloning

As for transposons /REMI, can

screen many mutants

simultaneously in infected host

Requires subsequent mutational

analysis to verify role in

virulence, can miss virulence

factors also expressed in culture,

requires molecular genetic system

for pathogen

May miss traits involving more

than one gene, requires a non-

pathogenic relative and molecular

genetic system

Requires a non-pathogenic

relative and subsequent mutational

analysis to verify role in virulence

Knowledge about suspected

virulence usually required

Gene cloning laborious,

possibility of >1 mutation per

genome

“Hot spots” for insertions,

insertional mutagenesis system

required

As for transposons/REMI ,

pathogen must infect as mixed

population

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria was a useful tool to tag

pathogens in order to visualize, by fluorescence microscopy techniques, their infection behaviour

under in vivo conditions. In this respect, visualization of GFP-labelled organisms by confocal

laser scanning microscopy (CLSM) is an effective, fast, and non-invasive tool allowing

spatiotemporal analysis of pathogen- host interactions (Pliego-Prieto, 2007).

2.12 Molecular Biological aspects

Confocal laser scanning microscopy (CLSM) images of avocado roots infected with the highly

virulent isolate CH53- GFP demonstrated that fungal penetration of avocado roots occurs

simultaneously at several random sites, but it occurs preferentially in the crown region as well as

throughout the lenticels and in the junctions between epidermal cells. Not only were R. necatrix

hyphae observed invading the epidermal and cortical root cells, but they were also able to

penetrate the primary and secondary xylem.

According to the available information regarding the mechanism of infection of fruit tree roots

by R. necatrix. Plants infected by R. necatrix normally show both aerial and root

symptoms that arise as a consequence of damaged roots and the delivery of toxic

compounds inside the plant vascular system .The first symptom that can be observed on

larger infected root surfaces is the existence of white cottony mycelium and white or black

mycelial strands. Fungal invasion of young mulberry tree roots has been reported to take place

by boring and dissolving cork cell and, on rare occasions, by wedging them. Alternatively,

invasion of adult roots into the inner tissues appears to occur primarily through the suberized

closing layers of the lenticels, generally as hyphal strands (Pliego et al., 2012). Invasion extends

through the cambium and wood to the trunk. On woody plants, the fungus is located

between the bark and the wood, developing very typical white mycelial fans (Fig. 2C–E).

Later, the white mycelium turns greenish-grey or black, and the pathogen forms indefinite

plaques within the bark and loosely aggregated strands of associated hyphae, which invade

the whole root system, causing a general rotting. Subsequently, the roots acquire a dark

brown colour. Infected trees do not always show aerial symptoms, making the diagnosis of

this fungus extremely difficult. The evolution of the symptoms expressed by the aerial

system,especially on fruit trees, can occur either quickly or slowly. In the first case, and in

a very short period of time, infected trees may suddenly decline in vigour, leaves wilt and

dry and, finally, trees eventually die. In the second case, symptoms develop more slowly

and, consequently, retarded growth can be observed with infected trees. Sparse foliage

may be observed in these trees, together with wilting of the leaves, chlorosis and the death

of twigs, branches and leaves. These symptoms worsen every year, when moisture and

temperature are favourable, and trees eventually die (Guillaumin et al., 1982) (Fig 2.4).

Fig 2.4: Root infection course of Rosellinia necatrix in avocado. This study was

conducted used the green fluorescent protein (GFP)-tagged R. necatrix

CH53 strain and visualized by confocal laser spectroscopy and scanning

electron microscopy. The whole process takes around 24 days in 1-year-

old avocado plantlets. The root infection by R. necatrix initiates with

fungal development on the root surface without penetration. Then,

root penetration can occur in different ways, including natural

openings, surface wounds and by direct penetration. Once inside the

root, R. necatrix spreads to the inner parts, resulting in xylem invasion,

as well as underneath the bark, leading to full symptom expression, as

shown in Fig. 2 (Pliego et al., 2012).

2.12.1 Double stranded RNA virus and Hypovirulence

In filamentous fungi, the presence of double stranded RNA (dsRNA) elements has been reported.

The elements are known to reduce the virulence of phytogenic fungi. Such dsRNA elements in

R. necatrix have also been reported. It has been hypothesized in 2005 by Ikeda et al that the

hypovirulent isolates were more likely to persist in soil as saprobes and thus attempts were

made to isolate these hypovirulent factors from soil. Belonging to eight different mycelial

compatibility groups (MCGs), sixteen isolates were obtained from two active and one

abandoned pear orchards in Japan. Out of these eight MCGs two were obtained exclusively

from soil. Other than these two isolates, isolates within the same MCG were similar in virulence,

competitive saprophytic ability (CSA) and mycelia growth rate wether or not they carried

dsRNA. Hypovirulence, weakened CSA and restricted mycelia growth on nutrient-rich media

these were the symptoms which were obtained from the other two isolates obtained from the soil

having multiple dsRNA segments. Ikeda et al observed that this isolates obtained from soil

contained various dsRNAs (44%) including hypovirulence factors, more frequently than the

isolates from the diseased roots in the same field (25%). Further he suggested that the isolation

of R. necatrix is an effective method to obtain isolates with dsRNAs, including hypovirulence

factors.

2.12.2 Characterization of Hypovirulent factors: W8, W370 and W779

Out of 1000 isolates of R. necatrix obtained from Japan for the identification of hypovirulence

factors many fungal isolates containing diverse types of dsRNA which were assumed to be

Mycovirus genomes were also observed. Virus containing isolates of R. necatrix W8 and W370,

showed irregular colony morphology and low virulence (Arakawa et al., 2002, Ikeda et al.,

2004).

One of these strain of R. necatrix W8 harbours four dsRNAs (L1, L2, M1, and M2, named

according to size). Out of these four M dsRNAs have been identified as the genome of

partitivirus Rosellinia necatrix partitivirus 1-W8 (RnPV1-W8), however the dsRNAs are thought

to belong to a distinct virus. The partivirus RnPV1-W8 has isometric particles with a diameter of

25nm comprising two genomic dsRNAs (2,299 and 2,279 bp).

In contrast the isolate of Rosellinia necatrix W370 harbored double-shelled, spherical particles

80 nm in diameter comprising equimolar amounts of 12 segmented genomic dsRNAs of 943 to

4,143 bp (Osaki et al., 2002; Wei et al., 2003; Wei et al., 2004). Osaki and coworkers obtained

12 segments from the hypovirulent strain of Rosellinia necatrix W370 and eight full length

cDNA sequences. All of them confirmed to be the member of the family Reoviridae as all the

eight sequences had conserved regions at the 5’ and 3’ termini. Morphological as well as

genomic analysis of this virus particle indicated that the virus was a novel reovirus designated as

R. necatrix Mycoreovirus 3 or RnMyRV3/ W370 (MyRV3). MyRV3, along with two

Cryphonectria parasitica Mycoreovirus spp. (MyRV1 and MyRV2), was placed in a newly

established genus Mycoreovirus in the family Reoviridae (Mertens et al., 2005). There is reduced

virulence of host fungus R. necatrix when get infected with MyRV3 in addition to the altered

colony morphology (Kanematsu et al., 2004). Likewise MyRV1 and MyRV2 infections also

cause hypovirulence in C. parasititca (Enebak et al., 1994; Hillman et al., 2004).

The complete nucleotide sequences of W370 dsRNA genome segments 1, 2, 3 & 5 were reported

in 2003/04 by Wei et al. The complete nucleotide sequence of the genome segment 1 encoded a

putative RNA-dependent RNA polymerase (RDRP). With a long open reading (ORF) and 47%

GC content, the nucleotide sequence of the genome segment 1 was 4143 bases long. Designated

as VP1, the deduced polypeptide contained 1360 amino acid residues (29-4110) with a predicted

molecular mass of about 153kDa. It showed some identity to the members of genera Fijivirus

and Cypovirus in the genus Coltivirus, these viral proteins belong to the Colorado tick fever

virus (CTFV) and European Eyach virus (EYAV). With a 3773 bases a single long ORF of

segment 2 encoded 1226 amino acid residues with a predicted molecular mass of approximately

138.5kDa. the nucleotide sequence of the segment 5 was 2089 bases long with a single long

ORF, whose deduced polypeptide contained 646 amino acid residues, with predicted molecular

mass of about 72 kDa.

On the other hand, the nucleotide sequence of the genome segment 3 was 3310 bases long and

has a GC content a little more of about 48.6% with a long ORF. Designated as VP3, the deduced

polypeptide contained 1086 amino acid residues (bases: 10-3270) with a predicted molecular

mass of about 121.9 KDa, showing no similarity to the other viral proteins.

Another isolate of Rosellinia necatrix W779 was isolated from soil in Ibaraki Japan (Ikeda et al.,

2005). Later on in 2009 Chiba and coworkers isolated particles ~50 nm in diameter from strain

W779 consisted of two dsRNA elements approximately 9 and 7 kbp termed as dsRNA-1 & 2 & a

major protein of 135 kDa encoded by the ORFs on dsRNA-1 (Chiba et al., 2009). It was also

observed that the purified virus particles were contagious and conferred hypovirulence on

vegetatively incompatible fungal strains. Sharing the conserved termini sequences at both the

ends, both possessed extremely long (~1.6 kb) 5’ untranslated regions (UTRs) similar to each

other, two ORFs, and relatively short 3’ UTRs. Though 3’-proximal ORF of dsRNA-1 encoded

RdRp showing low levels of sequence identity to those of members of the families Totiviridae

and Chrysoviridae. Phylogenetic analysis revealed that the W779 virus was to be placed into a

separate clade from the recognized virus families. These attributes indicated that dsRNA-1 and 2

represent the genome segments of a novel bipartite virus, designated Rosellinia necatrix

megabirnavirus 1 (RnMBV1), with virolocontrol agent potential. The establishment of a new

family, Megabirnaviridae, to accommodate RnMBV1 as the type species was proposed.

Two novel quadripartite dsRNA virus strains were identified namely Rosellinia necatrix

quadrivirus 1 strain (W1075) (Lin et al., 2012a) and Rosellinia necatrix quadrivirus 1 strain

W1118 (Lin et al., 2012b). Both quadriviruses posses quadripartite genome structure with a size

range of 4.9 – 3.7 kbp, each possessing a single large ORF, spherical particle morphology,

sequence homogeneity in the extreme terminal ends, 72-82% sequence identity between the

corresponding proteins and are only able to cause latent infection to R. necatrix.

After inoculating an apple orchard with two incompatible R. necatrix isolates strain W563 (virus

free, MCG 139) and NW10 (virus infected, MCG442). Yaegashi et al.,2012 recovered forty two

sub isolates of R. necatrix after 2-3 years and found that all are genetically identical to W563 or

NW10. However, 22 of them contained novel dsRNA. Six novel dsRNA (S1-S6) were found in

which S1 was a new victorivirus; S2, S3 and S4 were new partitiviruses and S5 and S6 were

novel viruses that could not be assigned to any known mycovirus family. Chiba and cowerkers

isolated a phytopathogenic strain of R. necatrix infected with a novel victorivirus named

Rosellinia necatrix victorivirus 1 (RnVV1) and a partitivirus. RnVV1 showed moderate level of

CP and RdRp sequence identity (34-58%) with other members of genus victorivirus (Chiba et

al., 2013).

2.13. Tools to study pathogenesis

Valuable knowledge about the molecular basis of the pathogenicity of R. necatrix could be

gained from genetic studies such as gene insertional mutagenesis. However, understanding the

genetic basis of its pathogenicity has been limited by the lack of a suitable transformation

system. Agrobacterium tumefaciens mediated transformation (ATMT), which has long been a

workhorse in plant science, has been exploited for fungal transformation. A. tumefaciens has the

ability to deliver its T DNA into chromosomes of the budding yeast, Saccharomyces cerevisiae

and diverse filamentous fungi. Besides ascomycetes and basidiomycetes, this technique has been

successfully applied to transform zygomycetes also. In comparison with Restriction Enzyme

Mediated Integration (REMI), ATMT does not require protoplasts and allows a broad spectrum

of starting material to be transformed. Protoplasts, hyphae, spores and even blocks of mushroom

mycelia tissues were transformed through ATMT with a higher efficiency. DNA transfer from A.

tumefaciens has been used for both gene knockout and gene transformation studies in

filamentous fungi and is being developed as a system for insertional mutagenesis in filamentous

fungi (Kano et al., 2011).

A number of transformation systems have been developed for filamentous fungi, including plant

pathogens. Kanematsu and co-workers (2004) reported the transformation of R. necatrix

protoplasts with plasmids pSH75 (Kimura and Tsuge, 1993) and pAN7.1 (Punt et al., 1987). In

addition, Agrobacterium tumefaciens-mediated transformation of R. necatrix has also been

reported (Aimi et al., 2005). Pleigo et al., 2009 generated a plasmid vector (pCPXHY1eGFP)

constitutively expressing EGFP and developed a protoplast transformation protocol.

Historically random mutagenesis studies have been effective for investigations of complex

biological processes such as pathogenicity. A method for generating non-homologous integration

events, termed restriction-enzyme-mediated integration (REMI), has been successfully used for

mutagenesis in a variety of organisms including plant pathogenic fungi (Kahmann and Basse

1999). Insertion mutagenesis by restriction enzyme-mediated integration (REMI) is a method

that is being used to generate mutations whose molecular basis can be easily identified (Shuster

et al.,1999). REM1 was first demonstrated in Saccharomyces cerevisiae, an organism in which

homologous integration is extremely efficient. Insertion mutagenesis has many attractive

features; most importantly, the mutation of interest is physically marked, providing a facile

means to isolate and analyze DNA flanking the site of the insertion (Schiestl et al., 1991).

Kuspa and Loomis (1992) first exploited REMI to generate tagged insertion mutations in

Dictyostelium discoidium. Introduction of various restriction enzymes with transforming plasmid

DNA whose free ends matched the accompanying enzyme stimulated transformation 20-60 fold.

Greater than 70% of the integration events were conservative integrations. In this random

insertional method, a restriction enzyme is introduced into the transformation mix. By a process

that is poorly understood, the enzyme gains access to the genomic DNA in the nucleus of the

transformation recipient and introduces double-stranded breaks at its recognition sites. These

breaks are recombinogenic with the transforming plasmid DNA that has been linearized with the

same restriction enzyme. In theory, integration of plasmid DNA into a gene at a restriction site

will cause a mutation, and that mutation will be tagged by the integrated plasmid. The success of

these transformation events depended on linearization of the transforming plasmid because no

integrations were seen with undigested plasmids lacking recognition sites for these restriction

enzymes. If the undigested plasmid contained a recognition site for the enzyme, a low frequency

of integration was seen, suggesting that digestion of both plasmid DNA and host genome during

the transformation process is less efficient (Riggle et al., 1998). The REMI technique is similar

to transposon tagging techniques that are available for other organisms and offers some of the

same advantages. A restriction enzyme that does not cut within the integrated plasmid can be

used to rescue it, along with some of the flanking genomic DNA, creating a plasmid that will

replicate in Escherichia coli. The flanking DNA can be sequenced and used to isolate cosmids or

plasmids from a library, and the rescued plasmid can be introduced back into the recipient

organism where homologous recombination should recreate the original mutant phenotype,

confirming that the tagged gene is important for a given function (Fig. 5).

Restriction enzyme mediated integration (REMI) has been used to clone pathogenecity genes

along with the identification of genes through an insertional mutagenesis approach. If the

comparison is drawn with PEG/CaCl2 mediated ttransformation, both REMI and ATMT can

improve transformation rates and frequency of single copy integrationevents, features desirable

for the recovery of insertional mutants (Roger et al., 2004).

Fig 2.5: REMI transformation and plasmid rescue. A. The REMI

plasmid (RP) contains a fungal promoter to drive transcription of a

fungal transformation marker. The bacterial transformation marker,

often the gene conferring ampicillin resistance,is needed for plasmid

rescue. Ideally, the REMI plasmid lacks homology to the genome of the

fungal recipient. B. The REMI plasmid (circular or linearized) is

transformed in the presence of a restriction enzyme (e.g., BamHI)

resulting in cleavage of the transforming plasmid and at respective

chromosomal sites. C. Free ends of cleaved plasmid and genome join

together. D. Plasmid rescue is achieved by plasmid excision from

genomic DNA together with flanking sequences (e.g., by using Mlu I)

and circularization with DNA ligase prior to subsequent transformation

in E. coli (Kahmann et al., 1999).

There are many advantages to REMI transformation. Transformation efficiencies are often

elevated by the REMI protocol. In addition, utilization of REMI for the successful generation of

mutants and gene isolation has been shown in many species including Dictyostelium discoideum

(Kuspa and Loomis 1992, 1994), Neurospora crassa (Kang and Metzenberg 1993), Ustilago

maydis (Bolker et al., 1995), Alternaria alternata (Akamatsu et al., 1997), Candida albicans

(Brown et al., 1995), Coprinus cinereus (Granado et al., 1997), and Magnaporthe grisea (Shi et

al., 1995; Sweigard et al., 1998).

The technique of REMI has been exploited in a variety of fungi ranging from hemi-acomycetes

and acomycetes to basidiomycetes. In most of the cases, development of REMI has involved

addition of restriction enzyme and the appropriate REMI vector to preexisting transformation

protocols, though optimization and choice of restriction enzyme for REM1 in various fungi is

somewhat empirical. As discussed previously the use of REMI as means to undertake genetic

analysis in fungi provides several advantages: the creation of random insertion mutations that are

physically tagged; the stimulation of transformation frequency several fold; and the creation of

single genomic insertions that are stable and unrearranged. In addition to insertion mutagenesis,

REMI is also being utilized in new ways such as for RFLP mapping, promoter trapping, and

dominant genetics; and this list of uses will undoubtedly grow. Transformation methods for

human pathogens including Histoplasma capsulatum, Cryptococcus neoformans, Aspergilllus

fumigattus, and Blastomyces dermatitidis, have been developed, allowing introduction of DNA

and, in some cases, homologous recombination (Worsham et al., 1990, Hogan et al., 1997).

REMI will likely be a useful additional tool for genetic analysis in these interesting and

important organisms (Table 2).

2.14 Marker genes for transformation

A wide range of genes have been found to be suitable as selectable markers for fungi. The hph

gene (hygromycin B resistance) is the most commonly used selection system because of its

effectiveness in most, but not all, systems. Other selective agents such as phleomycin,

sulfonylurea, nourseothricin, bialophos, carboxin, blasticidin S and benomyl have also been used

(Weld et al., 2006).

An alternative to drug resistance genes for transformation of fungi is to use auxotrophic markers

such as pyrG (a homologue of the Saccharomyces cerevisiae ura3 gene). Mutants that lack pyrG

are auxotrophic for uracil so vectors containing pyrG allow selection on uracil-deficient media.

Additionally, pyrG-deficient mutants are resistant to 5-fluoro-orotic acid (5FOA) which is toxic

in prototrophs. Positive/negative selection genes such as pyrG provide the possibility of

conducting a series of sequential transformation by using Blaster cassettes (Schiestl and Petes,

1991).

Table 2.2 Examples of genetic analysis in fungi using REMI (Riggle et al., 1998).

Organism Form of

transforming

DNA

Homology of

transforming

DNA

Restriction

enzyme

Stimulation of

transformation

Type of

integration

References

Candida

albicans

(imperfect)

Linear Some

homology to

genome

BamHl

-17 >95%

conservative

integrations

all appear

random

Riggle et

al.,1997;

Winegar et al

., 1989

Cochliobolus

heterosporus

(ascomycete)

Linear No homology

to genome

HinDIll - -20 Two tox-

mutants

analyzed

were

conservative

integrations

at

independent

HinDIll sites

Lyngholm et

al., 1994

Coprinus

cinereus

(basidiomycete)

Circular or

Linear

Homology BamHI,

EcoRI, Pstl

Kpnl (for

conservative

REMls)

BamHl

-7 32-67%

conservative

integrations

1201 all

appear

random

Granado et al.,

1997

Magnaporthe

grisea

(ascomycete)

Linear No homology

to genome

BamHI,

Bg/ll,

HinDIll,

EcoRV

<10 28-72%

conservative

integratrons

all appear

random

Shi et al.,

1995

Saccharomyces

cerevisiae

(hemi-

ascomycete)

Linear No homology

to genome

BarnHI, Bgl

II, (for

conservative

REMIs)

-7 60-90%

conservative

integrations

all appear

random

Schiest et al.,

1991,

Manivasakam

et al.,1998

Ustilago

maydis

(basidiomycete)

Cicular No homology

to genome

Bam HI None -50%

conservative

integrations

Balker et al.,

1995

2.15 From genomics to proteomics

Increasing interest have been developing in looking beyond the genome i.e investigating the

functions and interactions of the proteins themselves. For studying genome recourses are

developing to study proteome, by using recombinant DNA approaches to fluorescently label

endogenous proteins (e.g. GFP fusion proteins) or for epitope-tagging (translational fusion of an

antigenic determinant to the protein of interest). Combining annotated sequence information with

the method of targeted DNA integration achieved through homologous recombination, a library

of yeast with an epitope-tag integrated at the C-terminus of nearly every open reading frame has

been created. Although it is difficult to produce large-scale epitope-tagged libraries for any

filamentous fungi, though targeted epitope-tagging using high-throughput PCR methods have

been achieved in at least one filamentous fungus (Yang et al., 2004). However one of the

possilbility is to use High-throughput immunoprecipitation experiments with epitope-tagged

libraries to look beyond transcription profiles and directly monitor protein location, post-

transcriptional modifications and interactions with other proteins. Further, by creating fusions

with fluorescent proteins such as GFP and applying advanced techniques such as fluorescence

resonance energy transfer it is now possible to detect specific conformational changes within

proteins (Schultz and Boyle, 2005). In future we may see tagged proteins become widely used to

study biochemical reactions in vivo, in real time.

2.17 Induced resistance in plants

Specific biotic or chemical response can be produced by a plant which is basically the enhanced

defensive capacity of a plant to external stimuli, which was defined as induced resistance

(Bakker et al.,2007). However the more refined definition of the induced resistance was given by

Loon in 1997, “that a plant once appropriately stimulated, exhibits an enhanced resistance upon

‘challenge’ inoculation with a pathogen”.

Another general definition was given by Kloepper et al., 1992. According to him “it is a process

of active resistance dependent on the host plant’s physical or chemical barriers, activated by

biotic or abiotic agents (inducing agents)’. The term ‘induced resistance’ is not entirely

unambiguous. It might seem to imply that resistance was absent, but became present as a result

of the action of an inducing agent.

Resistance, according to Agrios (1988) is the ability of an organism to exclude or overcome,

completely or in some degree, the effect of a pathogen or other damaging factor. Netherlands and

J. W. Kloepper in Auburn, AL, discovered independently that induced systemic resistance (ISR)

is a mode of action of plant growth-promoting rhizobacteria (PGPR), especially fluorescent

pseudomonads, in suppressing diseases (Bakker et al., 2007).

In the last decade it has become clear that elicitation of ISR (induced systemic resistance) is a

widespread phenomenon. ISR is phenotypically similar to systemic acquired resistance (SAR)

that is triggered by necrotizing pathogens in that disease caused by a challenging pathogen is

reduced. This suggested that the range of pathogens that are controlled might be extended when

ISR and SAR are combined. Interestingly, when ISR and SAR are activated simultaneously,

enhanced disease suppression occurs against pathogens against which both Salicylic Acid and

ethylene responses are effective, such as P. syringae pv. Tomato. Thus, there appears to be room

to increase the effectiveness of induced resistance.

One of the most challenging research areas in the context of plant productivity improvement is

disease control of roots. The concept of manipulating the rhizosphere in such a way that

beneficial microorganisms with antagonistic and/or eliciting properties are favored would protect

roots from the deleterious effect of soilborne pathogens. Support for this concept came from the

discovery of suppressive soils in which the active microflora naturally controls the disease-

causing activities of pathogen populations. Soils naturally suppressive to fusarium wilt, pythium

damping-off, and Thielaviopsis basicola and Gaeumannomyces graminis-incited diseases have

been reported in different areas of the world and have been the focus of intensive studies dealing

mainly with the identification of the physicochemical and biological factors involved in

biological control.

Although a number of possibilities have been raised, for the disease control including microbial

competition for nutrients, competition for infection sites and root colonization, and, more re-

cently, plant-induced resistance through the rapid stimulation of a general cascade of nonspecific

defense responses, including structural barrier and pathogenesis-related proteins such as

chitinases and β-1,3-glucanases (Benhamou et al., 2002).

Over the last few years, a number of approaches, including chemical, biological, and microbial

treatments, have been proposed for the control of root borne disease. However, the value of these

options in agriculture and horticulture has yet to be realized, mainly because serious limitations,

such as phytotoxicity and variability, have delayed any transfer of technology from the

laboratory to the field. It is because of such difficulties, integrated pest management strategies,

involving more than one biocontrol product or agent, offer bright opportunities for success,

provided that the mode of action of each component is understood.

2.18 Industrial Applications of R. necatrix

Lignin is a structurally complex aromatic biopolymer which is recalcitrant to

degradation. The degradation of lignin is an important step in the mineralization of

carbon in nature. It has been demonstrated that the extracellular enzymes of some

white rot fungi are able to degrade lignin extensively. These extracellular enzymes

include lignin peroxidases, manganese peroxidases, and laccases. In addition to their

industrial application in delignification, laccases are also known to polymerize

phenolic compounds. This characteristic of laccases makes them of potential

interest for industrial applications involving the polymerization of phenolics in

liquids, the oxidation of dyes and dye precursors, and polymerization of lignin and

lignosulfonates. In fungi, besides a role in delignification, laccases appear to be

involved in sporulation, pigment production, and plant pathogenesis (Yaver et al.,

1996).