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REVIEW OF LITERATURE

The literature has been reviewed on the host- poplar, nursery diseases on poplars in

general and Bipolaris leaf blight in particular. Due to lack of direct information on the

disease, parallels from similar pathogen (other species of Bipolaris), host (other clones,

species of P. deltoides and related genus) and production system (agriculture) has been

drawn.

2.1. The Host: Poplar

The prime objective of forestry is to develop and protect forests, which are one of the

world’s renewable natural resources for maximum productive and protective functions.

Poplar is amongst world’s fastest growing multipurpose tree species. It is widely

distributed in many parts of the world and India. Poplars are known to naturally occur

in subtropical broadleaved hill forests, wet temperate, moist temperate, deciduous

forests and dry temperate forests. Poplars grow well on low lying and moist areas

preferring loamy soils but may be planted on riverbeds with sandy soils and in areas

with clayey loams in forest soils (Tiwari, 1993).

Poplars, by virtue of their fast growth, offer a great potential for meeting the

requirements of the farmers and wood-based industry in the country. Besides, they are

also widely accepted as industrial raw material, compatible with agriculture crops,

environmental ameliorating properties and short rotations of 6-12 years (Tiwari, 1993).

In India, poplars have become integral to agriculture, which is demonstrated by their

ubiquitous presence in the farms throughout the north. Poplars are usually raised as

single clone monocultures and, thus, prone to epidemic spread of pests. Poplars are

propagated vegetatively by use of cuttings in order to maintain their genetic purity.

Such cuttings may carry pests and diseases on them from one region to another and,

thus, may contribute to the spread of pests to new localities (www.fao.org, 17/8/2008).

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2.1.1 Taxonomic position P. deltoides

Kingdom Plantae

Sub Kingdom Tracheobionta

Super-division Spermatophyta

Division Magnoliopsida

Class Magnoliopsida

Sub class Dilleniidae

Order Salicales

Family Saliaceace

Genus Populus

Species deltoides

Source: Natural Resources Conservation Service (United States Department of Agriculture), 12/2/2009. 2.1.2. Distribution

Important countries, where poplar planting is initiated to meet the challenge of

increasing demand and shortage of wood supply are Belgium, France, Germany, Hungry,

Romania, Spain, Yugoslavia and Korea. In the east region, Afghanistan, Iran, Iraq, Israel,

Lebanon, Syria and Turkey also took up poplar rising. Australia and New Zealand too

attempted introduction of poplars successfully (Dalal & Trigotra, 1983).

Poplars are mainly restricted to the north Indian states. The researchers, after

trials of different species of poplars in hills as well as plains, were convinced that only P.

yunnanensis and P. deltoides were the most promising and successful species (Seth,

1969; Lohani, 1976; Chaturvedi, 1982). Forest resources in Punjab and Haryana are

almost negligible. Agriculture is the main land use. Therefore, poplar has a possibility of

growing as agroforestry species. Plantations of poplar (Populus spp.) are becoming

popular in the plains of northern India particularly in Haryana, Punjab, Uttarakhand and

Uttar Pradesh (U.P.) due to its fast growing habit, compatibility with crops, eco-friendly

and multipurpose uses in different industries.

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Under irrigated conditions of Punjab, Punjab Agriculture University (PAU) has

conducted a series of nursery (Sidhu, 1996) and field trials (Sidhu & Dhillon 2007; Singh

et al., 2008; Dhillon et al 2010; Dhillon & Sidhu 2010). In 1996, PAU released seven

clones i.e. PL-1(L-39/84), PL-2 (L-71/84), PL-3 (L-154/84), PL-4 (L-313/85), PL-5 (G-

48), PL-6 (L-188/84) and PL-7 (113324) on basis of field trials conducted at two

sites of the state from 1992 to 1996. The trials showed 26 to 35 per cent volume

production superiority than control (G-3). On the basis of four clonal trials in different

climatic zones, two clones (L-47/88 and L-48/89) were recommended during 2009. In

Haryana, promising clones reported are IC and G-3 (Singh et al., 1983; Dalal & Trigotra,

1983). Toky et al. (1996) reported CP-82-4-1 and CP-82-4-2 clones of P. deltoides better

than G-3.

Although, almost all clones of P. deltoides growing successfully in particular area

are suitable for agro forestry. However, Karanatak et al. (1994) have suggested some

clones (A-26, A-343, T-75, T-94, T-185, 73/53-3, 3276, 3294 and 3297) which are more

favorable for this purpose as they shed their leaves before the starting of sowing period

of agriculture crop and flush after harvesting. However, they cautioned that the clones

were suitable to Dehradun or similar climate. Dhiman and Gandhi (2006) analyzed the

match splint quality of wood of 8 clones and concluded that wood of clones G-48 and

Bahar produced match splints with low wood defect parameters i.e. crookedness and

cross grain splints. Total splint defects were higher in S7C15, Wimco-22, L-34, L-49 and

Udai clones.

For North West plains of U.P., suitable clones are G-3, G-48, D-121, D-61, D-67,

S7C8 and S7C15 (all from Americans origins; Piare Lal, 1991). Rawat et al (2001)

screened 75 clones at nursery stage in eastern U.P. and selected eleven best clones on

basis of independent culling method of selection. Another study (Singh et al., 2001)

conducted in eastern U.P. (Sultanpur) found significant variation in all growth traits,

number of branches and crown width among 50 clones with high expected genetic gain

of 30.28 per cent for volume by selecting 5 best clones (40-N, UD 9116, 25-N, 63-N and

UDH 1002).

Under sub-humid conditions of eastern Madhya Pradesh (now Chhattisgarh)

Puri et al. (2002) identified 19 promising clones out of 106 on basis of survival and

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growth performance in nursery trials. After four year of field evaluation they selected

five clones (65/27, S7C1, G-48, G-3 & D-121).

2.1.3. Characteristics of P. deltoides

It is also known as eastern cottonwood. It retains height up 18-32m and trunk diameter

0.5-1.0m. Trunk is broadly rounded or irregular, spreading and with some drooping

branches. The trunk of open grown trees often splits into more stems. Bark thin, smooth

and gray on young trees, becoming thick, rough and furrowed with age. Twigs stout,

yellowish green, gray or tan somewhat angled. Buds are yellowish brown, 1.25cm long,

slender, three-sided long pointed and resinous. Terminal buds are 1-2cm long. Leafs are

alternate on 5-10cm long flattened yellowish leaf stalks green and shiny above paler

beneath 6-12.5cm long nearly as wide broadly triangular with a straight or slightly

heart shaped base and abruptly pointed tip, coarsely toothed with gland tipped teeth,

thick and firm. In autumn, the leaves turn yellow. Flowers either male or female, borne

on separate trees in hairy bract catkins. Male catkins short stalked, reddish 8-10cm long

and densely flowered. Female catkins short stalked, greenish yellow 15-20cm long and

few flowered. Fruit are two- to four-valved capsule, borne in 15 - 25 cm long drooping

catkins, short-stalked, greenish brown, and elliptic. Seeds light brown with cottony hairs

attached. (www.vplants.org, 12/2/2009).

2.1.4. Cultivation

P. deltoides is the only species of polar that is planted on a significant scale in India. P.

deltoides, hereafter called poplar, constitutes the backbone of agro forestry in irrigated

plains of Northern India (Kishwan & Kumar, 2003). It has been estimated that 60,000

hectares equivalent plantations of poplar exists in India.

When poplar is planted on the field boundaries, kharif as well as rabi crops can

be grown in field throughout the rotation of poplar. In block plantation of poplar, the

usual kharif crops can be grown for two years only; thereafter shade-bearing crops like

ginger, turmeric, etc. are planted. However, rabi crops can be grown as usual. Poplars

also serve as a windbreak. The earliest contribution of poplar trees to the farmers is fuel

wood from pruning. Intercropping is almost always preferred as it provides agricultural

returns on the one hand and results in increased growth rate of poplar on the other due

to frequent irrigation and hoeing operations of agricultural crops. Pure poplar is seldom

raised. lf raised pure, the spacing is kept about 3m x 3m and the stems remain thin

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which fetch low price in the market. The rotation suggested for poplar cultivation in

India is 10 to 12 years but farmers prefer to cut it at 6 to 8 years rotation. Unlike

developed countries, coppicing of poplar at cutting cycles of 3 to 4 years is not practiced

in India. The felled areas are replanted with fresh nursery stock (Kishwan & Kumar,

2003).

Irrigation should be provided as soon as the planting of cuttings in any bed is

completed. Undue delay in irrigating the beds can cause dehydration of the planted

cuttings and result in poor sprouting. The first irrigation should be medium heavy, so

that, about 5 to 7 cm water is uniformly above ground level at the time of irrigation.

Subsequent irrigation should be light and the interval may vary between 7 to 10 days

depending upon the type of soil. Light sandy soils will need frequent irrigations whereas

soils with clay need irrigation at longer intervals. Proper and effective drainage of

excess water during rainy reason is essential to prevent lodging and collar rot. After the

rainy season, one to two irrigations per month will be adequate

(www.eucalyptusclones.com/downloads/agro_pdf9, 17/8/2011).

Well decomposed farmyard manure which is rich in macro- as well as micro-

nutrients essential for the plants should be applied to the total area under poplars while

preparing the land for inter-cultivation of rabi and kharif crops. Application of

nitrogenous, phosphatic and potassic fertilizers as well as micro nutrients will depend

on the fertility status of the land. At least, during the first year of the plantation

micronutrient may be arranged to guard against any possible deficiency

(www.eucalyptusclones.com/downloads/agro.pdf, 17/8/2011).

2.1.5. Utilization

2.1.5.1. As a tree

P. deltoides has been found suitable as line support for overhead power and

telecommunication lines. It is suitable for the manufacture of match splints and

plywood. Young plant of P. deltoides was evaluated for making fibers hardboards

(Tewari, 1993). The wood of poplar is used in papermaking, plywood, matchsticks,

packing cases, sport goods, light construction and furniture.

Poplar was introduced in tarai region of U.P. to meet the need for the plywood

and matchwood industries (Chaturvedi & Rawat, 1994). However, large scale plantation

of poplar has started only during the last decade and thousands of hectares of fertile

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land in north India have been put under plantation of poplar in the state of U.P.,

Uttarakhand, Haryana, Punjab and Himachal Pradesh. As a result of increased poplar

plantation, plywood industries have been mushroomed in this region of India.

2.1.5.2. As Intercrop

Poplar is deciduous tree and very suitable for agro forestry system. It has no shading

effect on crop, rather adds to soil fertility through its leaf litter. Environmental

amelioration is another intangible benefit from poplar planting. It has been estimated

that poplar grows 36 times faster than other Indian forest trees and, therefore, utilizes

36 times more carbon dioxide (Chandra, 1998).

Growing auxiliary crops with poplar is highly beneficial to the tree crop. Its

growth has been enhanced up to 40 percent. This is because of regular irrigation

weeding, hoeing and fertilizer application during cultivation of the secondary crops (Jha

& Gupta, 1991). Intercropping of cereals, cash crops forage, etc. add to the income

through agriculture. Wheat, oat, sorghum, maize, sugarcane, berseem, turmeric, zinger,

potato, dhanicha, etc. can easily be grown as inter-crops. Intercropping with poplar is

remunerative during the rabi as well as kharif season.

Farmers grow poplar both in block as well as boundary plantation along with

agricultural crops. The latest trends are to plants them on agriculture field

simultaneously with companion crops. This is a sustainable land management system,

which increases the yield of the land by combined production of crops and forest plants.

There are evidences to prove that tree planting improves the agro climatic condition

and mitigates their adverse effect by changing the microclimatic in area. Some

progressive farmers have obtained three times higher income from poplar and

agricultural crop combination than pure agriculture (Mandal et al., 2005). Therefore,

poplar agro forestry is undoubtedly very lucrative business.

This is considered to be the best agro-forestry species for inter-cropping, having

high rate of growth, short rotation, good economic returns and has less effect on

intercrops but proper management of intercropping is necessary. Farmer’s plant this

species on their cropland and within 6-7 years harvest and market it to the industry.

During the kharif season soybean, maize and grain legume can be grown while during

the Rabi season wheat, potato, peas, etc., can grow successfully. Poplar is winter

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decideous and adds tremendous amount of leaf litter to the soil (www.tribuneindia.com,

2010).

2.1.6. Marketing and Economics

Poplar wood is sold by weight. Profits to the tune of 38.8 and 100.9 percent of

investments are reported from rising of nursery stock within one year (Singh &

Vashista, 2001). Benefit:cost ratio of 1.92:1 and 2.13:1 have been estimated with pure

poplar and with poplar + intercropping in a pay-back period of 7 years (Dillon et al.,

2001). Owing to very little risks and high profits in poplar cultivation, large farmers and

absentee land-lords prefer to put their lands under poplar-based agro forestry rather

than other agriculture/agro forestry options (Kishwan & Kumar, 2003).

Poplar (P. deltoides) is extensively grown agroforestry tree in selected district of

Punjab, Haryana, Uttar Pradesh and Uttarakhand. Approximately 2 core sapling planted

per annum throughout the poplar growing region has created verdure sylvan landscape

in agricultural land. These saplings quickly grow, form thick forest and attain 10 percent

crown cover for both block (400 trees/ha) and boundary plantation (one ha base) in the

first year itself (Dhiman, 2009) and qualify as forest based on the crown parameter used

by the Forest Survey of India (FSI, 2009) much faster than other tree species grown

both inside and outside farms than other forests.

2.2. Nursery Diseases of Poplars in India

Many fungi, bacteria, viruses, etc. are capable of causing damage to leaves, branches,

boles and roots of the poplars. The Table2.2.1. details about the diseases of poplar in the

country.

Table2.2.1. Nursery diseases of poplars in India

Disease Pathogen Distribution Reference

Nursery disease

Set rot

Botryodiplodia.

palmarum

Lasiodiplodia

palmarum

U.P. Singh and Khan (1979)

B. palmarum Solan (H.P.) and U.P. Singh and Khan (1983)

B. palmarum H.P. Khan (1988)

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B. palmarum Lalkua Forest Research

Nursery, Lalkua,

Nainital

Harsh and Kumar (1992)

B. palmarum and

Macrophoma sp.

Northern India Pandey and Khan (1992)

B. palmarum U.P. and H.P. Khan (1999)

Leaf rust Melampsora

rostrupii

Oidium sp. and

Myrothecium

roridum

H.P. and Kashmir valley Singh and Khan (1979)

Leaf spot Cercospora

populina,

Sphaceloma

populina,

H.P. and Kashmir valley Singh and Khan (1979)

Leaf rust

and Powdery

mildew

Melampsora sp.

and

Uncinula sp.

J. & K. and H.P. Rehill and Puri (1980)

Leaf spot Cercospora sp.

S. populina,

M. roridum,

Septoria populi

and Phyllosticta

adjuncta

J. & K. and H.P. Rehill and Puri (1980)

Leaf rust Melampsora sp. J. & K. Singh and Khan (1983)

Exotic Rust M. ciliata Himalayas Singh and Khan (1983)

Alternaria tip

blight and

Cladosporium

leaf spot

Alternaria stage of

Pleospora

infectoria and

Cladosporium

humlie

J. & K. and nurseries of

U.P.

Singh and Khan (1983)

Melampsora rust Melampsora sp. Kashmir valley Singh et al. (1983)

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Melampsora rust M. ciliata H.P., U.P. and J&K Khan et al. (1988)

Leaf rust and

Powdery mildew

M. populina and

U. salacis

H.P. Khan (1988)

Leaf spots and

Asteroma leaf

bilght

S. populi, C. humile

and Asteroma

frondicola

H.P. Khan (1988)

Alternaria tip

blight and

Phyllosticta leaf

spot

Alternaria stage of

P. infectoria and P.

adjuncta

H.P. Khan (1988)

Pollaccia blight,

Melampsora rust

and Powdery

mildew

Pollacia elegans,

M. cilata,

M. populina and

U. salacis

Kashmir valley Rehill et al. (1988)

Septoria leaf

spot,

Cladosporium

leaf spot and

Sphaceloma leaf

spot

S. populi,

C. humile and

S. populina

H.P. and J. & K. valley Rehill et al. (1988) and

Sharma and Sharma

(2000)

Pollacia blights P. nigra

P. elegans

Potusai, Kamraj Forest

Division, J & K State

Khan and Mishra (1989)

Cladosporium

leaf spot

C. humile H.P. Khan (1990)

Leaf rust M. ciliata H.P. Khan (1990)

Leaf spot C. humile,

Alternaria sp. and

Phyllosticta sp.

H.P. Khan (1990)

Sclerotium leaf

spot

Sclerotium rolfsii New Forest, Dehradun Mishra and Khan (1991)

Leaf blight Bipolaris maydis U.P. (Tarai), Punjab and

Haryana

Chauhan and Pandey

(1992)

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Leaf spot Alternaria

alternata, P.

adjuncta,

S. populi and S.

rolfsii

U.P., Punjab and

Haryana

Singh et al. (1991)

Leaf rust M. ciliata and

M. larici- populina

U.P. , H.P. and J.&K. Pandey and Khan (1992)

Leaf spot A. alternata, P.

adjuncta, S. populi,

S. rolfsii M.

roridium and

Dreshclera maydis

U.P. , H.P. and J.&K. Pandey and Khan (1992)

Bipolaris leaf

blight

B. maydis U.P., Punjab and

Haryana

Chauhan and Pandey

(1995)

Leaf rust Phomopsis sp.,

R. solani,

M. ciliata and

M. larici-populina

U.P. and H.P.

U.P. and H.P.

U.P. , H.P. and J.&K.

Khan (1999)

Leaf disease C. humile,

D. maydis,

Alternaria sp. and

S. rolfsii

Eastern Himalayas,

Payal, Haryana and

Central Tarai Forest

Division, U.P.

Khan (1999)

Alternaria leaf

bilght

A. alternata H.P. Sharma et al. (1999)

Leaf rust M. ciliata H.P. Sharma and Sharma

(2000)

Leaf spot Cladosporium sp. Solan, Kullu, Lahaul &

Spiti and Kinnaur

Sharma and Sharma

(2000)

Leaf spot Alternaria sp.,

Phyllosticta sp.

FRI campus, Dehradun Pandey (2002)

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and Bipolaris sp.

Leaf rust M. populina

Europe, China, India and

Chile

Pai and Mc.Cracken

(2005)

Alternaria leaf

spot and tip

blight

Curvularia leaf

spot

A. alternata and

Curvularia lunata

H.P., J. & K., Uttarakhand

and Punjab

Sharma ( 2009)

Stem rot S. rolfsii New Forest, Dehradun Mishra and Khan (1991)

Stem rot Phellinus

phephyphloccus,

Phellinus noxius

Tarai plains of U.P. Khan (1999)

Root rot Ganoderma

lucidum

Lacchiwala Range,

Dehradun, East Forest

Division, U.P.

Singh and Khan (1979)

Root rot G. lucidium Phillaur, Punjab Rehill and Puri (1980)

Root rot

G. lucidum Tarai Central Forest

Division, Srinagar

Singh and Khan (1983)

Root rot

G. lucidum Tarai plains, U.P. Khan (1999)

White root rot Rosselinia

nectrarix

H.P. Khan (1988)

White root rot Macrophoma sp. U.P. and H.P. Khan (1999)

White root rot

Dematophora

necatrix

H. P. Sharma (2009)

Plantation diseases

Pink disease and

Canker

Corticum

salmonicolor and

B. palmarum

Haldwani and Lalkuan

plantations, U.P

Singh and Khan (1979)

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Pink disease,

Canker,

Dieback and

Heart rot

C. salmonicolar,

B. palmarum,

Phomopsis sp. and

Trametes sp.

Pipalparao, Belachaur

and Gangapur Patia

plantations, U.P.

Tarai & Bhabar

Plantation Division, U.P.

Rehill and Puri (1980)

Poria root rot Poria vinta Tanda Block, T. & B.

Plantation Division,

Dehradun, U.P.

Rehill and Puri (1980)

Canker and

Die back

C. salmonicolor

and

Cytospora

chrysosperma

U.P. and H.P. Khan (1999)

2.3. Poplar Blight

2.3.1. Historical perspective

The blight pathogen has been identified as a major parasite of poplars and clones like G-

3, G-48, D-153 and 6244 showed moderate to high susceptibility in nurseries (Jones &

Lal, 1989). It has been detected in its conidial stage as Bipolaris maydis on a number of

clones of P. deltoides causing serious foliage blight (Tewari, 1993). Later, a leaf blight of

P. deltoides caused by B. maydis was reported by Chauhan & Pandey (1992).

In 1995, Chauhan and Pandey (1995) identified isolates of B. maydis to be race T

on the basis of their virulence to certain male cultivars of G-3 of host from Texas

provenance raised in agro forestry system by M/S Wimco Limited in India particularly

in the states of Uttar Pradesh (north–west region), Punjab and Haryana. Besides, toxin

production, sclerotia formation, light pigmentation and poor sporulating potency on the

culture media under controlled condition were also studied. Among diseases, leaf blight

caused by B. maydis was the most serious disease. However, it affects only G-3 clone and

cultivation of this clone in the affected areas has been stopped (Kishwan & Kumar,

2003). B. maydis leaf blight, a serious disease on P. deltoides, has been reported by

Sharma (2009) to be prevalent in Uttarakhand and Punjab.

Bipolaris maydis (Nisikado) Subram. et Jain [= Bipolaris maydis (Nisikado et

Miyake) Shoem.], the anamorph of Cochliobolus heterostrophus (Drechsler) Drechsler

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(family-Pleosporaceae, order- Dothideales, phylum- Ascomycota), long known as an

important leaf parasite on maize, recently observed in India (isolates designated T race)

on some clones - mainly male of Texan origin - of P. deltoides, where it is responsible for

a leaf blight of very high incidence; on the contrary, tests showed that P. nigra and P.

ciliata are totally resistant (www.fao.org, 2/2/2008.)

2.3.2. Blight in nursery trials

Reliable, time- series information is available in the annual reports of Wimco Seedlings

Ltd., Baghwala, Rudrapur regarding disease status of Bipolaris blight vis-a- vis genotype

susceptibility. Mainly two fungal diseases were noticed in poplar germplasm nursery.

The leaf blight caused by B. maydis and leaf blotch caused by C. populina usually

appeared after onset of monsoon. Poplar clone, G-3 was heavily infected by these

diseases;, highly susceptible to leaf blight, clone G-3 is becoming unsuitable for planting

out in humid location (Annual Report, 1997). Approximately 36,000 half- sib seedlings

of 1996 population poplar were produced. Of these, initially, 188 individuals were

selected for multiplication based on their high growth rate and resistance to B. maydis

(Annual Report, 1998). It was also observed that clone G-3 was highly susceptible

whereas other clones were resistant against B. maydis.

In the Annual Report of 1999, it was mentioned that germplasm bank of 284

poplar clones consisting of 100 developed by Wimco Seedlings at R & D centre

Baghwala, Rudrapur, 73 of University of Forestry & Horticulture, Solan, 12 of U. P.

Forest Department and 97 exotics were maintained in Baghwala. Only for 10 cuttings of

each clone, diameter and height/diameter ratio were recorded at 12 month age. Only 2

clones were recorded growing above population mean. Clone WSL- 54 attained

maximum average height of 481cm and average collar diameter of 3.54cm. A poplar

germplasm bank consisting of 379 clones (226 of Wimco seedlings Limited, 64 of

University of Forestry & Horticulture, Solan, 8 of U. P. Forest Department and 81

exotics) was maintained in nursery trials at spacing of 70cm x 60cm at Research &

Development Centre, Baghwala, Rudrapur during 2006-07 (Annual Report, 2007).

Three hundred forty seven clones were retained out of 2006 poplar germplasm by

culling 3 clones on account of their susceptibility to leaf blight diseases and poor growth

whereas, 32 new clones were added to the germplasm bank during the year. It was

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found that only clone G-3 was highly susceptible for B. maydis while, remaining clones

planted in germplasm were found resistance to the disease.

2.3.3. Symtomatology

Chauhan and Pandey (1992) defined the symptoms on poplar in detail i.e. diseases

appear after onset of the monsoon usually during July. Pink to light brown lesion of

pinhead size develops on leaves. Lesions gradually convert into dark brown rounded

spot, often surrounded by a chlorotic zone having concentric ring towards center. B.

maydis was the causal agent of the disease. In rainy or humid weather, blight often

appears by collapsing together of several lesions or when lesions involve midrib and/or

large veins of the leaves. Chlorosis frequently extends well beyond the necrotic lesions

and subsequently turns into big blighted patches. Sometimes, light infected leaves get

complete chlorosis. Severely infected/chlorotic leaves curl in dry weather and

ultimately fall off prematurely. Necrotic lesions, sometimes developing into canker, also

appear on green shoots of the susceptible clones. Premature defoliation of transplant,

trees of the susceptible clones predisposes them to weak parasites/saprophytes and

environmental stresses resulting extensive dieback. Tewari (1993) reported the disease

in late September as irregular blotches starting from the margin of leaves. They turned

grayish brown in the centre surrounded, sometimes, with a pale halo. Under cool and

humid conditions, the blotches covered the larger area of the leaf bearing grayish black

specks consisting of conidiophores and conidia all over the dried portion. In advance

stage, the leaves of the entire plant up to crown region exhibit as if they were scorched

by fire.

Young lesions of maydis leaf blight on maize are small and diamond shaped. As

they mature, they elongate. Lesions may coalesce, producing a complete ‘burning’ of the

leaves. They vary in size and shape among inbreeds and hybrids with different genetic

background. Race ‘O’ produced tan, elongated (2-6 x 3-22mm) lesion between the vein

with limited margins, with buff to brown borders. The exact leaf blight symptoms on

southern corn depend upon the race of the agent and the strain of corn affected. As a

general rule, tan lesions are seen on leaves with the number and size depending upon

the fungal race and the strain of corn. In the worst case the lesions are numerous and

can be several centimeters long and have dark red or purple edges. Ears are also

infected with a black substance that is actually masses of conidia (asexual spores) on

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kernels that can lead to ear and cob rot. Stalks may also be damaged

(www.cbwinfo.com, 9/12/2011).

First report of leaf spot caused by B. seteriae was on the leaves of cassava

(Manihot esculenta) in China. Initially elliptical, chlorotic, and water-immersion lesions

of 2 to 4 mm in diameter appeared. These lesions became dry and yellow due to the

progress of the disease. A brown halo was around the lesions, and in wet conditions, a

dark gray mildew often appeared in the middle of the lesion. Diseased leaves turned

yellow and the plants eventually became defoliated (Shi et al., 2010).

2.3.4. Taxonomic position

Kingdom Fungi

Phylum Ascomycota

Class Euascomycetes

Order Pleosporales

Family Pleosporaceae

Genus Bipolaris

2.3.5. Causal agent Bipolaris is a dematiaceous, filamentous fungus. It is cosmopolitan in nature and is

isolated from plant debris and soil. The pathogenic species have known teleomorphic

states in the genus Cochliobolus and produce ascospores. The genus Bipolaris contains

[Type text] Page 34

several species. Among these, three well-known pathogenic species are B. spicifera, B.

australiensis and B. hawaiiensis.

2.3.6. Cultural and morphological features

Table2.3.6.1. Fig.2.3.6.1. to 2.3.6.3. detail colony and spore characters of those Bipolaris

species that have been/are being reported from P. deltoides.

2.3.7. Pathogenicity

The pathogenicity of B. spicifera was tested on the leaves of two cultivars of watermelon

(Peacock 124 and Mabrouka) which was widely cultivated in Morocco. The infection

coefficients (incidence × severity index) of the cv. Mabrouka and Peacock 124 after

inoculation with B. spicifera conidial suspensions were 53.3 and 22.4, respectively.

Calculated disease development rates were greater for cv. Peacock 124 than for cv.

Mabrouka. Conidium production of B. spicifera on inoculated leaves was very abundant

on the two cultivars, and the fungus was re-isolated from lesions on inoculated plants.

This is the first record of B. spicifera on watermelon in Morocco (Mhadri et al., 2009).

The artificial inoculation of the healthy leaves of three plants of Punica granatum

L. ’Nana’ by conidial suspension of the pathogen induced the same lesions to that

observed in nature by Kadri (2011). The diseased foliar surface and the total number of

diseased leaves 30 days after inoculation with B. spicifera conidial suspension were 37.5

and 78 percent. Conidia production of B. spicifera on inoculated leaves was 0.81 x 105

spore cm-2 and the fungus was re-isolated from lesions on inoculated plants. This is the

first report of B. spicifera on pomegranate in Morocco.

The pathogen B. seteriae was isolated and pathogenicity was established by

following Koch's postulates on Cassava (Manihot esculenta Crantz). This is an important

food crop in tropical regions of China (Shi et al., 2010). Young, healthy and fully

expanded green leaves of Cassava cv. HuaNan205 were surface sterilized and then

inoculated by spraying them with a suspension of conidia (1 × 105 conidia per ml) of the

isolate. Sterile water was used as a control. The leaves were kept in a humid chamber at

28°C for 4 days, in the mean time similar symptoms were observed on the leaves. The

pathogen was re isolated from inoculated leaves

[Type text] Page 35

2.3.6.1. Features of Bipolaris species

Causal organism and

host

Hyphae/

Culture type

and color

Conidiophore Conidium Reference

B. maydis

Graminecous family

Populus deltoides

Septate and

brown

Initally white to

grayish brown

and become

olive green to

black;

Velvetty to

wooly

Conidiophores (4.5-6 µm wide) are brown,

simple or branched, geniculate and sympodial,

bending at the points where each conidium

arises from. This property leads to the zigzag

appearance of the conidiophore.

The conidia, which are also called poroconidia, are

3- to 6-celled, fusoid to cylindrical in shape, light

to dark brown in color and have sympodial

geniculate growth pattern. The poroconidium (30-

35 x 11-13.5 µm) is distoseptate and has a

scarcely protuberant, darkly pigmented hilum.

This basal scar indicates the point of attachment

to the conidiophore. From the terminal cell of the

conidium, germ tubes may develop and elongate

in the direction of longitudinal axis of the

conidium

www.doctorfungus.org,

11/10/2010

B. spicifera

Vigna aconitifolia

Graminicolous species

common on plant

material particularly

grasses

Septate and dark

Initally white

soon become

dark gray and

olivaceous black

texture;

Wooly to

cottony

Conidiophores may be up to 300 µm in length,

are sympodial, geniculate, simple or branched,

bearing conidia through pores or openings

(poroconidia).

Conidia contain predominately 3 transverse

distosepta or pseudosepta (septa that do not

extend to the cell wall with cells enclosed within

sacs) and 4 cells. They measure approximately 20-

40 x 9-14 µm. A flattened hilum or point of

attachment is seen on the basal cell. Conidia

germinate from both poles (bipolar)

www.cybertruffles retrieved

on 9/12/2011

www.doctorfungus.org,

retrieved on 11/2/2011

B. setariae

Brachiaria reptans ,

Panicum fasciculatum,

Pennisetum typhoides

Setaria italica , and

Septate and dark

brown

Initally brown to

grayish black;

Flocosse to

Conidiophores solitary or in small groups,

straight or flexous, sometimes geniculate, pale

to mid brown or olivaceous brown, up to 200

µm long, 5-9 µm wide, sometimes swollen at

the base to 11 µm wide.

Conidia slightly curved or sometimes straight,

fusiform or navicular, pale to mid golden brown,

smooth, with 5-10 pseudosepta, (45-) 50-70 (-

100) x (10-) 12-14(-15) µm

(Sawada) Shoemaker, 1959

www.cybertruffles,

9/12/2011

www.mycobank.com,

[Type text] Page 36

other cereals. It also

isolated from soil and

leguminous seeds

cottony 11/2/2011

B. setariae.

Desmostachya

bipinnata, Oryza sativa

Sorghum sp.

Triticum sp. (India)

lawn grass and

gramineous crops

Conidiophores were fasciculate and brown,

septate and straight and the basal cell was

enlarged and hemispheric

al.

Well-developed conidia were long- obclavate,

obtuse at both ends, straight, brown, with five to

eight transverse septa, and measured 49.7 to

117.1 × 13.3 to 17.2 μm

Sivanesan, (1987)

www.cybertruffles,

9/12/2011

Shi et al. (2010)

[Type text] Page 37

Fig.2.3.6.1. Detailed structures of conidiophores and

condia of B. maydis.,

Source: www.cybertruffle.com.

[Type text] Page 38

Fig.2.3.6.2. Detailed structures of conidiophores and conidia of B. spicifera. Original in: Hoog, G.S. de. 2000, Atlas of clinical fungi, ed. 2: 1-1126.

[Type text] Page 39

Fig.2.3.6.3. Detailed structure of conidia and

bipolar germination of B. seteriae.

Source:www.cybertruffle.com.

[Type text] Page 40

2.3.8. Host Range

The hosts of different Bipolaris spp. are as below:

Causal organism Host Location Reference

B. spicifera Seeds of bermuda

grass

Seoul Branch Station

of National Plant

Quarantine Services

of Korea located at

Kimpo airport

Koo (2004).

B. spicifera Isolated from

necrotic leaves of

watermelon

Taroudant area of

southern Morocco

Mhadri et al. (2009).

B. spicifera Leaves of 100 plants

of pomegranate

(Punica granatum L.

’Nana’)

Public gardens and

the University of

Sciences of Kenitra

city (Morocco)

Kadri (2011)

D. seteriae

(B. seteriae)

Pearl millet seed India Shetty et al. (1982)

and Ahmed and

Reddy (1993)

B. seteriae

and B. incurvata

Diseased orchids,

bromeliads, proteas,

and other plants.

Heliconia in Hawaii Sewake and Uchida

(1995).

B. seteriae

and B. incurvata

Grasses Heliconia fields in

Hawaii

Uchida and Aragaki

(1995).

B. seteriae Cassava

(Manihot esculenta

Crantz)

Danzhou, Hainan

Province and

tropical regions of

China

Shi et al. (2010)

2.3.9. Nutrition

Fungi have quite simple nutritional requirements. They need a source of organic

nutrients to supply their energy and to supply carbon skeletons for cellular synthesis.

But, given a simple energy source such as glucose, many fungi can synthesize all their

other cellular components from inorganic sources – ammonium or nitrate ions,

phosphate ions and trace levels of other minerals such as calcium, potassium,

[Type text] Page 41

magnesium and iron. Fungi that normally grow in a host environment or in other

nutrient-rich substrates might require additional components, but still, the nutrient

requirements of most fungi are quite simple. Having said this, fungi need to capture

nutrients from their surroundings. The cell wall prevents fungi from engulfing food

particles, so fungi absorb simple, soluble nutrients through the wall and plasma membrane. In

many cases, this is achieved by releasing enzymes to degrade complex polymers and,

then, absorbing the nutrients released by these depolymerase enzymes. Fungi produce a

huge range of these enzymes, to degrade different types of polymer. In fact, there is

hardly any naturally occurring organic compound that cannot be utilized as a nutrient

source by one fungus or another (Deacon, 2006).

Five strains of H. maydis Nisik. et Miyake and two strains of H. carbonum were

grown on 14 carbohydrates and 13 fatty acids each as the sole carbon source. All

carbohydrates except L (-) -sorbose supported excellent growth. Fatty acids having 12

or more carbon atoms supported growth of all strains except H. carbonum Race II,

which failed to grow on linolenic acid. Optimum growth of strain NRRL 5128 H. maydis

Race T in shaken culture occurred within 7 days at 280C. Under these conditions, this

strain utilized 45-55 percent of corn oil when incorporated into the medium at 5

percent by volume as the sole carbon source (Ellis, 1973).

Silver scurf, caused by Helminthosporium solani, is an important storage disease

of potatoes. Experiments designed to evaluate control alternatives are limited by

difficulty in producing conidial inoculum. In an effort to better understand how this

difficulty could be overcome this study evaluated the influence of various carbon-to-

nitrogen (C: N) ratios, carbon concentrations, and amino acids on conidial germination,

colony diameter and conidiation of H. solani grown on solid-phase basal salts media.

Under the conditions tested, the highest concentrations of conidia were produced with

1.25 to 2.5 g of carbon/L at a C: N ratio of 10: 1. Higher C: N ratios or higher carbon

concentrations reduced conidiation. Total conidia production was improved by use of

tyrosine or arginine as the sole nitrogen source. Use of leucine, lysine, methionine,

phenylalanine, or threonine severely inhibited H. solani conidia production. Use of a

nitrogen source containing a mixture of amino acids resulted in a defined medium that

permitted conidiation and growth of H. solani that was similar to or better than that

obtained with standard V8 Juice medium (Elson, 1998).

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Bipolaris Shoemaker, Drechslera Ito and Exserohilum Leonard and Suggs are

known to be pathogenic to food crops, foliage and turf grasses (Yamaguchi, 2010). A

selective culture medium for fungal isolation will be useful for studies of diseases

caused by the fungi. The sensitivity of Bipolaris, Drechslera and Exserohilum to 9 kinds of

carbon sources were tested in vitro. While D-mannose, a carbon source, enhanced the

growth of those fungi, especially it was effective in growing Bipolaris and Drechslera

that grew a little bit slowly on potato sucrose agar. A selective medium has been

developed based on potato extract broth containing D-mannose (1%), agar (1.5%),

thiophanatemethyl (100mgL-1), and chloramphenicol (100mgL-1) as an antibiotic, and

the pH was adjusted to 4.8. Plant pathogenic fungi, Bipolaris, Drechslera and Exserohilum

were consistently isolated from diseased rice, oat and red sprangletop, respectively by

using the selective medium.

The EF-37 isolate, one of DSE fungi (Dark -septate endophytes), is beneficial to

the growth and development of its host plant, Saussurea involucrata Kar. et Kir. The

cultivation requirements including basic culture medium, temperature, light, pH, carbon

source and nitrogen compounds were studied for their effects on mycelial growth of a

dark-septate endophytic (DSE) fungus EF-37 by using one-factor-at-a-time method.

Potato dextrose agar (PDA) was the best medium for the growth of endophyte EF-37.

Our studies showed that 20°C, 24 h dark cultivation and pH 7 significantly influenced

the growth of endophyte EF-37 on PDA medium. Moreover, glucose and calcium nitrate

were found to be the best nutrients for EF-37 growth. Under the optimal cultivation

conditions, DSE fungus EF-37 isolate could grow actively. This is the first study about

the effect of cultivation conditions on the growth of this strain, which provides the

preparatory knowledge for the biological characteristics of DSE fungus EF-37. (Ya-li Lv

et al., 2010)

Sulphur is also one of the important constituents of a suitable nutrient medium

for fungi, but it is required in much smaller quantity in comparison to other essential

elements. It plays a vital role in their metabolism; it enters into the composition of the

mycelium and the spores of many fungi. It also takes part in protein synthesis,

respiration and other biochemical processes. Volkonsky (1933) classified fungi into two

categories on the basis of their sulphur requirements (I) Parathiotrophs- organisms

utilizing only reduced form of sulphur (II) Euthiotrophs- organisms utilizing sulphate

[Type text] Page 43

and other oxidised sulphur. The importance of sulphur compounds with special

reference to their role in biological methylation by fungi has been reviewed by

Challenger (1953). In spite of its importance there are certain fungi that grow without

sulphur (Steinberg, 1941; Srivastava, 1951; Agarwal, 1955). There is considerable

variation as regards the response of various fungi to sulphur compounds (Armstrong,

1921; Saksena et al., 1952; Agarwal, 1955; Agnihotri , 1962; Kumar,1962). Rastrick and

Vincent (1948) have made an extensive study on the utilization of sulphur compounds

and they reported that fungi converted essentially all the sulphate sulphur into organic

compounds. Among sulphur compounds, sulphates have mostly been reported to be

good sources for the growth of various fungi by several workers (Armstrong, 1921;

Mosheretal, 1936; Tandon, 1950). On the other hand, Volkonsky (1933 & 34) reported,

that members of Saprolegniaceae were unable to utilize sulphur as sulphate

(www.biologiezentrum, 7/10/2011).

2.3.10. Management

2.3.10.1. Chemical management

The genetic basis of susceptibility to southern corn leaf blight is well known and plants

resistant to it are widely available and constitute the first line of defense. If necessary,

fungicides can also be used against B. maydis. A large number are approved for use and

these include: chlorothalonil, mancozeb and propiconazole (www.cbwinfo.com,

21/10/2011). The best chemical management against maydis leaf blight of maize was

spraying of mancozeb@2g/l of water (www.agritech.tnau.ac.in, 9/12/2011).

Seed treatment was effective for controlling mortality due to seedling blight (B.

sorokiniana and Fusarium sp.). In growth stage trial of 1999-2000, seed treatment was

also effective for seedling mortality due to S. rolfsii. Percentage of black seed (carrier of

spot blotch for next season) was reduced with a single early spray (45 days after

sowing) for Tilt-250EC only and for combined treatment also, which suggests the

reduction of inoculum level of soil and seed borne B. sorokinana. Significant yield

increase of 31-48% was obtained for Tilt-250EC combined with Vitavax-200 in a single

early spray (35-45 days after sowing). This increase of yield for Tilt-250EC was only 22-

25% and for Vitavax-200 only 14-29%. The result suggests the reduction of inoculum

level of B. sorokiniana for seed, foliar, root and soil with combined treatment (Mehta &

Igarashi, 1985; Loughman et al., 1998). Dithane M-45 and Bayleton have been able to

[Type text] Page 44

inhibit growth of the Drechslera maydis in laboratory bioassay test in low concentration

(Khan, 1999).

Twelve seed sample of rice were tested by Ahmed et al. (2002) and all were

found infected with brown spot diseases caused by B. oryzae. Highest (5.5%) and lowest

(1.5%) incidence was found in sample Bhabokhali and Mahozompur, respectively. Four

fungicides viz. Bavistin, Hinosan, Tilt-250EC and Dithane M-45 were evaluated against

B. oryzae. Dithane M-45 was the best with 100% reduction of the prevalence of the

pathogen at 0.3% of the seed weight as the seed treatment and inhibited the mycelial

growth about 62.83% at 500ppm followed by Tilt-250EC (54.67%), Hinosan(29.60%)

and Bavistin (28.00%), respectively. All test fungicides were effective against B. oryzae

at higher concentration.

Overall, Medallion + Daconil provide good control as soon as 2 WAFT compared

to the other fungicide treatments. In addition, 2 WAFT all treatments showed significant

reductions in the number of Helminthosporium leaf spots (Drechslera sp. and Bipolaris

sp.) per unit area compared to the control, but it wasn’t until 6 WAFT that the other

fungicide treatments attained the same level of control as Medallion + Daconil. In

addition, Medallion + Daconil provided the best control throughout the 12 weeks of the

study at Washington State University Golf Course at Pullman,WA. Increased rates of

Medallion did not result in increased disease control. Daconil alone provided as good

control as any of the Medallion treatments alone (Golob & Johnston, 2004).

The sensitivity of Bipolaris, Drechslera and Exserohilum to 11 different fungicides

was tested in vitro by Yamaguchi (2010). Thiophanate-methyl, a chemical fungicide, had

little impact on the growth of B. oryzae (Breda de Haan) Shoemaker, D. avenacea

(Curtisex Cooke) Shoemaker, and E. rostratum (Drechsler) Leonard and Suggs. Chemical

fungicides examined were copper oxychloride (Sun-Bordeaux, 73.5% active ingredient),

benomyl (Benlate, 50% active ingredient), captan (Osocide, 80% active ingredient),

chinomethionat (Morestan, 25% active ingredient), maneb (M-Dipher, 75% active

ingredient), manzeb (Ziman Dithane, 75% active ingredient), polycarbamate (Bis-

Dithane, 85% active ingredient), polyoxins (Polyoxin AL, 10% active ingredient),

thiophanate-methyl (Topsin M, 70% active ingredient), potassium bicarbonate (Kari-

Geen, 37% active ingredient) and triflumizole (Trifmine, 30% active ingredient). All

fungicide formulations used in this study were wettable powders. B. oryzae, D. avenacea

[Type text] Page 45

and E. rostratum did not grow on the media containing 100ppm of maneb, manzeb,

polycarbamate and triflumizole. The fungal growth was mostly suppressed by

polyoxins. It was suggested that the Bipolaris, Drechslera and Exserohilum species were

sensitive to those fungicides. On the media containing 100ppm of benomyl, captan,

chinomethionat, copper oxychloride and potassium bicarbonate, the fungi grew to some

extents; however, the growth was slower compared to those on control media without

fungicides. On the other hand, the fungal growth was vigorous on the medium

containing thiophanate-methyl. The results revealed that Bipolaris, Drechslera and

Exserohilum were resistant to thiophanate-methyl at a concentration of 100ppm.

Therefore, thiophanate-methyl was suggested to be useful for the selective medium.

Moreover, as thiophanate- methyl has been reported to be effective against a wide

range of fungi including Monilia, Gloeosporium, Botrytis, Sclerotinia, Corticiium, Fusarium

and Pyricularia, this chemical fungicide is expected to suppress the growth of

saprophytic fungi on Gramineae such as oat, red sprangle top and rice plants.

Leaf spot is a disease provoked by the fungus B. maydis, which causes great

damages for annual crops such as corn, wheat and oats. In vitro tests were accomplished

to evaluate the efficiency of the mycelial growth inhibition of the pathogenic fungus

through five fungicides: tetraconazol, tebuconazole, azoxystrobin + cyproconazole,

trifloxystrobin + propiconazole and trifloxystrobin + cyproconazole (Yamashita, 2010).

For the determination of the efficiency of the products, the daily growth of the fungus

was evaluated, being compared with the control. All of the treatments were found

effective for the inhibition of the growth of the fungus.

2.3.10.2. Biological Management and Biogenic Interactions

Biological control by the antagonistic microorganism is a potential, non- chemical and

ecofriendly tool for crop protection against phyto-pathogenic fungi and the

management of several plant diseases (Papavizas, 1985). Considering the cost of

chemical pesticides and hazards involved, biological control of plant diseases is now

increasingly being practiced all over world. The use of antagonitic plant pathogens is

risk free when it results in enhancement of resident antagonist (Monte, 2001).

Following mixed inoculation with Cochliobolus sativus, incitant of spot blotch,

and Pyrenophora tritici-repentis, incitant of tan spot, wheat leaves developed less

necrosis than average produced by the two pathogens alone at inoculum concentrations

[Type text] Page 46

equal to those used in the mixed inocula. Spot blotch predominated over tan spot

following simultaneous inoculation or sequential inoculation where P. tritici-repentis

preceded C. sativus by up to 6hr. Antagonism occurred even when inocula contained

only 20% C. sativus. Inoculation with C. sativus resulted in reduced conidial germination,

slowed germ tube development and reduced appresorium formation in P. tritici-

repentis. Tan spot development may be suppressed in the field where environmental

conditions favour spot blotch C. sativus or its metabolites could potentially be

manipulated to produce an effective biological control for tan spot of wheat (Da Luz &

Bergstrom, 1987).

Black point is a brownish or black discolouration of wheat kernels and biological

control is a complementary strategy to manage the disease. This work evaluated the

effect of five strains of Trichoderma harzianum and one strain of T. koningii on the

growth of B. sorokiniana and A. alternata and compared the results of screening tests

under controlled conditions and field evaluations on bread and durum wheat ears.

Disease incidence, infection percentage and seedling emergence percentage determined

in a greenhouse assay were evaluated. Dual cultures showed that Trichoderma spp.

inhibited significantly the mycelial growth of B. sorokiniana between 36 and 71 percent

and of A. alternata between 41 and 61 percent. Microscopic examination of B.

sorokiniana and A. alternata showed plasmolysis and vacuolization of hyphae of the

pathogens in the presence of the antagonists tested. With pre-inoculation of wheat ears

at anthesis under field conditions, disease incidence, infection percentage by blotter

tests and seedling emergence in the greenhouse did not show significant differences

between controls and treatments with Trichoderma spp. (Monaco et al., 2004).

Interactions of 10 Fusarium spp., namely F. equiseti, F. longipes, F. moniliforme, F.

oxysporum, F. proliferatum, F. scirpi, F. pallidoroseum, F. sporotrichioides, F. solani and F.

subglutinans with other fungi viz., Alternaria alternata, Aspergillus niger, A. flavus, A.

terreus, A. versicolor, Cladosporium herbarum, Drechslera hawaiiensis, Paecilomyces sp.,

Penicillium digitatum, P. funiculosum, Rhizoctonia solani and T. hamatum were studied in

vitro by Fakhrunnisa (2006). In dual culture plate assays, T. hamatum showed inhibition

in growth of Fusarium spp., by producing zones of inhibition.

In order to assess the potential of rhizopheric micro organism in biological

control of soil-borne diseases, 180 isolates of both Pseudomonas and Bacillus spp. from

[Type text] Page 47

rhizoplane and surrounding soil of healthy and infected wheat were collected by

Soleimani et al. (2005) in Hamadan province Iran. Only 9 isolates with the high

antagonist ability against the growth of the two pathogenic fungal spp., B. australiensis

and B. sacchari were selected by dual culture method and purified.

Pathogenic micro-organisms living on wheat foliages may interact with each

others. This study was conducted to reveal the interactive relationships existing

between spot blotch causing fungus, B. sorokiniana and selected pathogenic fungi

subsisting on wheat foliages. Four fungal pathogens of wheat were selected and the

intensity and severity of the selected pathogenic fungi on wheat leaves were assessed.

Pure cultures of the fungi were produced by isolating them from the spot blotch

infected and blighted wheat leaves. Separate in vitro dual culture studies in completely

randomized design with five replications were carried out to assess the interactions

between each pair of B. sorokiniana and selected rival pathogens of wheat foliages.

Percent inhibition in radial growth of either fungus was calculated. Viability test of the

mycelium at the interface zone and pathogenicity test of the isolates were carried out. B.

sorokiniana strongly inhibited the colony growth of Cercospora sp. and Phoma sp. under

in vitro conditions. Similarly, there was no effect on colony growth of either B.

sorokiniana or Bipolaris sp. due to dual culture with each other. The dual culture of B.

sorokiniana and A. triticina results in the suppression of colony growth of both fungi.

There were non- significant differences in percent growth inhibition between the first

and the second week of dual culture in some of the tested fungi. The viability of mycelia

of all the tested fungi was intact in dual culture. B. sorokiniana exerts antagonistic ability

against some minor pathogens of wheat foliages under in vitro conditions (Bhandari &

Ranamukharachchi, 2010).

2.3.11. Toxin

In the fungal kingdom, the ability to cause disease in plants appears to have arisen

multiple times during evolution (Does & Rep, 2007). In many cases, the ability to infect

particular plant species depends on specific genes that distinguish virulent fungi from

their sometimes closely related non- virulent relatives. These genes encode host-

determining ‘virulence factors’ including small, secreted proteins and enzymes involved

in the synthesis of toxins. These virulence factors typically are involved in evolutionary

arms races between plants and pathogens. Current knowledge of these virulence factors

[Type text] Page 48

from several fungal species in terms of function, phylogenetic distribution, sequence

variation and genomic location have been summarized. Also some issues that are

relevant to the evolution of virulence in fungi toward plants; in particular, horizontal

gene transfer and the genomic organization of virulence genes have been addressed.

A toxin can be defined as microbial metabolites excreted or released by lysed cell

which in very low concentration is directly toxic to cells of the suspect. In pathology the

term toxin is used for a product of the pathogen, its host and pathogen –host interaction

which even at very low concentration directly acts on livings host protoplasm to

influence the course of disease development or symptoms expression. Pathotoxin

produce all or most of the essential symptoms of the diseases in susceptible host as a

convincing evidence of their causal role and can be host specific such as victorin, or non

specific. Only victorin meets fully the stricts requirements of a pathotoxin. For others

evidence of pathotoxicity is indirect and independent confirmation is lacking (Singh,

2009). Therefore, their status as pathotoxin is only tentative. Phytotoxin are the

substances for which causal role in disease is merely suspected rather than established.

These are product of parasites which induce few or none of the symptoms caused by the

living pathogen. They are non specific and there is no relationship between the toxin

production and pathogenicity of the diseases causing agent.

A host specific toxin is a metabolic product of a pathogenic micro organism

which is selectively toxic only to the susceptible host of the pathogen. These substances

have a very high degree of toxicity to the suspect in low concentrations at which they do

not affect other organism and they produce essential symptoms of the diseases when

placed in the healthy susceptible host (Singh, 2009).

Alternaria alternata tobacco pathotype, the causal organism of brown spot

diseases of tobacco produced a host selective toxin (named AT-toxin). The toxin was

purified from culture filtrates and this purified toxin inhibited the seedling growth of

both susceptible and moderately resistant cultivars of tobacco at 0.2µg/ml. To

determine the toxin activity following three bioassay procedures were employed. In a

leaf spray assay, sample solution to be tested were sprayed with atomizers on detached

leaf pieces (3 x 3cm) which were kept in moist chamber at 260C at florescent light. The

toxin activity was accessed by the development of characteristics necrotic spots and

chlorosis of leaf pieces treated with a series of test solution 24 to 48h after the onset of

[Type text] Page 49

treatment. In a leaf drop assay, detached pieces 3x3 cm were slightly wounded at center

with a needle and kept on sponge mat in moist chamber. The leaf, thus, prepared was

immediately treated by a drop (30µl) of sample solution to the wounded portion.

Necrosis and chlorosis developed on the leaves were recorded 48h after the treatment.

In the root dip assay, a seed of tobacco and other plants were pre-incubated on a wet

filter paper in Petri dishes at 260C. The uniformly germinated seed were transferred on

to filter paper 2cm in diameter in a small vial containing 0.5ml of sample solution. Root

length seedling was measured after 5 days of incubation at 260C. The average length

was compared to control (Kodama, 1990). Among several plants tested, only species

belonging to the genus, Nicotiana were sensitive to the toxin. These results show AT-

toxin has host recognition factor in the genus, Nicotiana - A. alternata pathosystem.

Culture filtrates of A. sonchi, its organic extract, the chromatographic fractions

and pure compounds 24-27 were assayed by leaf disc-puncture bioassay on S. arvensis

and a number of non-host plants (Punzo, 2009). The plants were produced from pieces

of underground shoots or seeds and grown in a greenhouse. The discs (10 mm diam.)

were cut off well-expanded leaves with cork borer, placed on moistened filter paper and

punctured by sharp needle in the centre. Crude organic extract, chromatographic

fractions and pure compounds were dissolved in a small amount of Et OH and then

brought up to desirable concentration with distilled H2O. The final concentration of Et

OH in test solutions was 5% v/v that is non toxic to leaves of all plants in the control.

Droplets (10μl) of the test solution were applied on the discs and, then, incubated in

transparent plastic boxes at 24°C under 12h photoperiod. After 2 days of incubation, the

diameter of the necrotic lesions (mm) was measured. Alternethanoxins A and B showed

a significant phytotoxin activity against host plants and several other weeds.

Alternethanoxins A and B didn’t show antimicrobial activity. Application of both

alternethanoxins on leaves of host plant did not showed synergistic effect.

Pathogenic reactions of total eleven isolates of B. maydis obtained from infected

leaves of poplar as well as maize were studied by Chauhan and Pandey (1995). Both

group of isolates caused leaf blight to their natural hosts- poplar or maize within 10-

15days of incubation. P-isolates caused flecking to the maize cultivar whereas, Z isolates

were non-pathogenic to the poplar cultivars. It was observed that P-isolates infected

young shoot of poplar cultivars while Z-isolates did not infect stalk tissues of the maize

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cultivars during the study period. Toxin metabolites were invariably isolated by

chloroform extraction from the culture filtrate of P-isolates, but not from Z-isolates.

Responses of the cultivars of the both poplar and maize were tested to the different

concentrations of the toxin dissolved in distilled water. It is obvious that sensitive

reaction to the toxin was exhibited by only poplar but not maize cultivars. Moreover,

toxin concentration was recorded as an important factor determining rate of necrosis.

Leaf discs floated on 500-1000ppm concentration exhibited about cent percent necrosis

within 12-15h of inoculation. At 100ppm concentration, necrosis was very slow

compared to the higher concentration. But at 10ppm only flecking was recorded in

compared to control (absolutely healthy).

Screening for resistant barley genotypes in response to fungal toxin of B.

sorokiniana was assessed on standing barley plants as well as selected callus lines

(Chand, 2008). For the standing lines, manifestation chlorosis in response to toxin

infiltration showed a significantly slower diseases progress as compared to the necrotic

lines. Also necrosis in the callus tissues of the susceptible cultivar in MS medium

supplemented with different concentrations of the crude toxin was significantly higher

than the callus tissues of the chlorotic lines. Similar host response to the toxin in in vitro

and field situation open up the possibility of screening barley cultivars for resistance to

spot blotch using callus culture as against classical methods of screening in order to

increase accuracy and save time and space.