Pak. J. Phytopathol., Vol. 25 (01). 95-100

95

Official publication of Pakistan Phytopathological Society

Pakistan Journal of Phytopathology ISSN: 1019-763X (Print), 2305-0284 (Online)

http://www.pakps.com

PATHOGENIC AND NON-PATHOGENIC FUNGI ISOLATED FROM SOIL AND ROOTS OF TOBACCO

Mubashar Raza*, Muhammad U. Ghazanfar, Muzammil Hussain, Zafar Iqbal, Yasir Iftikhar Department of Plant Pathology, University College of Agriculture, University of Sargodha, Pakistan.

A B S T R A C T

The study investigated the isolation of soil borne pathogenic fungi from soil and root samples of tobacco crop. A Soil dilution plate technique was used for isolation of fungi on Potato Dextrose Agar (PDA) media. Identification and characterization was done with the help of authentic manuals of fungi. A total 10 genera were isolated and identified which were parasitic, saprophytic and free-living fungi including pathogenic species Alternaria alternata, Fusarium oxysporum, Rhizoctonia solani, Phytophthora nicotianae and Trichoderma harzianum a free-living fungus. Incidence of Phytophthora nicotianae and Rhizoctonia solani was only seen in root and soil samples respectively whereas Alternaria alternata, Fusarium oxysporum and Trichoderma harzianum were found in both root and soil samples. Other isolated soil borne fungi included Aspergillus, Chaetomium, Geotrichum, Mucor and Penicillium. Occurrence of all these pathogenic fungi from root and soil samples was also calculated.

Keywords: Tobacco, pathogenic fungi, non-pathogenic fungi.

INTRODUCTION

The pathogenic micro-fungal floras of field soils cause

root rots, seedling damping-off and vascular wilts

diseases in plants (Lichtenzveig et al, 2006). Soil borne

pathogens are adapted to survive and grow in bulk soil

but rhizosphere is the playground where a fungus

establishes parasitic and symbiotic relationship with

plants (Raaijmakers et al, 2009). Soil borne fungal

pathogens infect number of plants. The most important

genera include Alternaria, Armillaria, Aspergillus,

Chaetomium, Cylindrocladium, Fusarium, Geotrichum,

Penicillium, Phytophthora, Pythium, Rhizoctonia and

Sclerotinia (Azaz, 2003). Trichoderma genus is also soil

borne but it is non-pathogenic to plants (Ahmad et al.,

1987). The species of Armillaria, Cylindrocladium,

Phytophthora, Pythium, Rhizoctonia cause root rot

diseases characterized by decay of true root system.

Stem, collar and head rots are caused by species of

Fusarium, Rhizoctonia, Sclerotina, Sclerotium,

Phytophthora and occasionally Aspergillus niger, all

produce symptoms of wilting and death of leaves as well

as whole plant. Species of Fusarium, Pythium,

Phytophthora, Rhizoctonia, and Sclerotium fungi affect

the seeds during germination, pre-emergence or post-

emergence phases of seedling establishment

(Lichtenzveig et al, 2006).Fungi in genus Trichoderma is

common in soil and root ecosystems act as a bio-control

agent against plant pathogens. It has been known since

at least the 1920s and recent study shows that they are

parasites of other fungi as well as opportunistic,

avirulent and plant symbionts (Harman et al, 2004 and

Harman, 2005).

T. koningii, T. lingorum and T. virens inhibit the growth

of Alternaria alternate in tobacco (Mandare et al., 2008).

It has been investigated that root rot effect in tobacco

seedling due to Rhizoctonia solani is reduced by

application T. harzianum (Gveroska and Ziberoski,

2011). Experiments conducted on several crops shows

that strains of Trichoderma reduces the disease effect

caused by Phytophthora palmivora, Rhizoctonia solani,

Fusarium spp., Sclerotium rolfsii and Pythium spp. (Tran,

2010). The aim of present investigation to isolate

pathogenic and non-pathogenic fungi from the

rhizosphere and root samples of tobacco to observe the

occurrence of soil fungi.

* Corresponding Author:

Email: mubasharraza73@yahoo.com

© 2013 Pak. J. Phytopathol. All rights reserved.

Pak. J. Phytopathol., Vol. 25 (01). 95-100

96

MATERIAL AND METHODS

Sample collection and study site: Soil and root

samples were taken from rhizospheres of tobacco with

the help of spatula to a depth of about 15 cm. The

spatula was applied perpendicular to the vertical

surface of the profile (Azaz, 2003). All samples were

collected in plastic bag from Wazirabad and different

sites of Mandi Bahaudin which were made airtight and

labeled as sample 1, 2, 3, 4, 5 and 6. Site 1 was of

Sodhran village that is tehsil of district Wazirabad and

site 2, 3, 4, 5 and 6 of Dera Beram, Mano Chalk and

Marala villages of Mandi Bahaudin. Two commercial

varieties of tobacco like SPEGTH-G-28 and K-399 were

grown on site 1, 5, 6 and 2, 3, 4 respectively (Table:1).

Isolation of Fungus from soil samples: Soil dilution

was prepared for the isolation of fungi from the soil and

roots of tobacco crops. To prepare soil dilution five

screw cap test tubes with 9 ml distilled water were

autoclaved and arranged 1-5 into the Laminar Flow

Hood for further processing. One gram soil sample

weighed and added to the first test tube shake

thoroughly by Vortex mixer to allow mixing of soil

sample and microorganisms, 10 fold serial dilutions

were prepared (Sharpley, 1960). Serial dilutions were

prepared from 10-1, 10-2, 10-3, 10-4 and 10-5. To obtaining

fungal colonies, 3rd, 4th and 5th dilution were used. One

milliliter (1ml) of solution from third dilution were

taken and spread on the media plates with the help of

spreader. After each spreading, spreader was sterilized

and this procedure was repeated for test tube 4th and

5th. Plates were placed in an incubator in inverted form

at 22±3 °C for 3 days after spreading (Arotupin, 2004).

Isolation of Fungus from root samples: Root samples

of tobacco crop firstly washed thoroughly under

running tap water for isolation of fungal endophytes

and 0.3 cm root samples were surface-sterilized in 3%

sodium hypochlorite or chlorox 1% with 0.03 Tween 20

for 1-3 minutes followed by rinsed thrice with sterilized

distilled water (Jose et al, 2012). Root were dried by

placing them between the layers of sterile blotter

papers and plated directly in 9 cm PDA Petri-dishes. The

plates were incubated at 22 ± 3 0C and examined after 7

days (Amusa, 2001).

Identification and purification of fungi: Mixture of

various soil borne fungi were grown on PDA medium

plates and a single spore of each fungi transferred to

fresh PDA medium plate with the help of sterilized

needle for further isolation and purification (Fang et al,

1983). The cultures were identified at genus level on the

basis of macroscopic (colonial morphology, color,

texture, shape, diameter and appearance of colony) and

microscopic characteristics (septation in mycelium,

presence of specific reproductive structures, shape and

structure of conidia, and presence of sterile mycelium

(Zafar et al., 2007) with available literature Barnett

(1998) and Bissett (1991 a,b,c).

Occurrence of Fungi: Occurrence of various fungi from

root and soil samples was calculated by following

formula (Iftikhar et al. 2010).

RESULTS AND DISCUSSION

Ten genera of fungi with several isolates were identified

from soil and root samples of tobacco include

Alternaria, Aspergillus, Chaetomium, Fusarium,

Geotrichum, Mucor, Penicillium, Phytophthora,

Rhizoctonia and Trichoderma. Most of the isolated fungi

belong to phylum Ascomycota, Mucor, Phytophthora,

Rhizoctonia belongs to Zygomycota, Oomycota and

Basidiomycota repectively. Both saprophytic and

parasitic mycoflora were isolated from root and soil

samples (Table: 2, 3). Aspergillus niger, Fusarium spp.

and Penicillium spp. were isolated from soil sample of

tobacco variety (SPEGHT-G-28) in Sodhran village of

Wazirabad. Fusarium spp. was reported to be

pathogenic on tobacco and cause Fusarium wilt

(Rodriguez, 2007). Alternaria alternata, Aspergillus spp.,

Chaetomium spp., Geotrichum spp., Mucor spp.,

Trichoderma harizanum, Rhizoctonia solani, Fusarium

spp. and Penicillium spp. were reported from soil

samples of different sites of Mandi Bahudin have

SPEGHT-G-28 and K-399 verities. Alternaria alternata

and Rhizoctonia solani are responsible for causing

brown spot and root rot disease of tobacco respectively

(Gveroska and Ziberoski, 2011).

The root samples collected from Wazirabad and Mandi

Bahudin were heavily infested and infected by

Alternaria alternate, Aspergillus niger, Geotricum spp.,

Mucor spp., Trichoderma harzianum, Rhizoctonia solani,

Fusarium spp. and Penicillium spp, and some extent of

Phytophthora spp.. Trichoderma harzianum acts as a

parasite of Alternaria alternata, Fusarium oxysporum,

Phytophthora nicotianae and Rhizoctonia solani (Tran,

2010., Gveroska and Ziberoski, 2011). Trichoderma

harzianum suppresses the growth of pathogenic fungi

through overgrowth or by formation of small tufts

Pak. J. Phytopathol., Vol. 25 (01). 95-100

97

A B

C D

D E

F G

H I

Isolated Fungi from Tobacco Field (A) Alternaria alternaria (B) Aspergillus spp. (C) Aspergillus flavous (D) Fusarium roseum(E) Fusarium oxysporum (F) Mucor spp. (G) Penicillum spp. (H) Phytophthpora spp. (I) Five days colony of Trichoderma harzianum (J) Fully developed colony of Trichoderma harzianum.

(Harman, 2006). Number of fungi isolated from flue-

cured and nonflue-cured leaves of tobacco which were

Alternaria, Aspergillus, Chaetomium, Cladosporium,

Epicoccum, Fusarium, Penicillium, Trichoderma and

Nigrospora (Welty et al., 1968).

In present study, fungal species of Aspergillus and

Fusarium were dominant in both soil and root samples

of tobacco rhizosphere. On the contrary, species of

Chaetomium, Geotrichum and Phytophothora present in

soil and roots samples respectively. Other pathogenic,

Pak. J. Phytopathol., Vol. 25 (01). 95-100

98

non-pathogenic and free living isolated fungi were

Alternaria alternata, Mucor spp., Penecillium spp.,

Rhizoctonia spp. and Trichoderma harzianum. English

and Mitchell (1988) reported that Aspergillus, Fusarium,

Penicillium and Trichoderma grow rapidly in

rhizosphere of tobacco.

Table 1: Detail of sites surveyed and fungi isolated.

Sites Sample no. Village Variety Fungi isolated from soil Fungi isolated from

roots

Wazirabad

1 Sodhran SPEGHT-G-28 Fusarium spp. Fusarium spp.,

Alternaria spp.

Mandi

Bahaudin

2 Dera Beram K-399 Fusarium spp.,

Trichoderma harzianum,

Alternaria spp.

Phytophthora spp.,

Fusarium spp,

Trichoderma harzianum,

Alternaria spp.

3

Mano chalk K-399 Fusarium oxysporum,

Trichoderma harzianum,

Rhizoctonia spp.,

-

4 Mano chalk K-399 Fusarium oxysporum,

Trichoderma harzianum

-

5 Marala SPEGHT-G-28 - Fusarium spp.,

6

Marala SPEGHT-G-28 - Fusarium spp.,

Trichoderma spp.,

Table 2: Presence of fungi in different sites of tobacco field soil.

Sample

Altern

aria

Asp

ergillu

s

Ch

aeto

miu

m

Fu

sariu

m

Geo

tricum

Mu

cor

Pen

ecillium

Rh

izocto

nia

Trich

od

erma

Sample 1 x A. niger x x x x x

Sample 2 x

Sample 3 x

Sample 4 x F. oxy x x

Table 3: Presence of fungi in roots of tobacco crop of different soil sites.

Sample

Altern

aria

Asp

ergillu

s

Fu

sariu

m

Mu

cor

Pen

ecillium

Ph

yto

ph

tho

r

a

Rh

izocto

nia

Trich

od

erma

Sample 1 R x x x

Sample 2 R x

Sample 5 R x x x x x

Sample 6 R x A. niger x x x

Table 4 (a): Occurrence (%) of various fungi from root and soil samples.

Sites Alternaria Aspergillus Chaetomium Fusarium Geotricum

- Soil Roots Soil Roots Soil Roots Soil Roots Soil Roots

Wazirabad 0 100 100 100 0 0 100 100 0 0

Mandi Bahudin 66.6 33.3 100 100 66.6 0 100 100 33.3 0

Pak. J. Phytopathol., Vol. 25 (01). 95-100

99

Table 4 (b): Occurrence (%) of various fungi from root and soil samples.

Sites Mucor Penicillium Phytophthora Rhizoctonia Trichoderma

- Soil Roots Soil Roots Soil Roots Soil Roots Soil Roots

Wazirabad 0 100 100 100 0 0 0 0 0 0

Mandi Bahuudin 100 66.6 100 33.3 0 33.3 33.3 33.3 100 66.6

Occurrence of Aspergillus and Fusarium at the sites of

Wazirabad and Mandi Bahudin regions was high on

both soil and root samples followed by Penicillium,

Mucor, Alternaria, Trichoderma, Rhizoctonia,

Chaetomium, Geotrichum and Phytophthora. Aspergillus

and Fusarium shows 100% prevalence from root and

soil samples of Wazirabad and Mandi Bahudin regions.

All detail regarding occurrence of various fungi isolated

from roots and soil samples is given in Table 4 (a,b).

Number of colonies of Aspergillus flavus, Aspergillus

fumigatus, Aspergillus niger, Aspergillus nidulans,

Aspergillus terreus, Penicillium chrysogenum, Penicillium

frequentans, Penicillium funiculosum, Trichoderma viride,

Trichoderma harzianum, Fusarium oxysporum, Fusarium

solani, Curvularia clavata, Curvularia lunata, and

Rhizopus stolonifer isolated per Petri from 1 gram soil

plate to give the percent contribution of each isolate in

different crops for example Paddy, Corn, Ragi, Red garm,

Cotton and Sugarcane (Gaddeyya, 2012). The results

obtained clearly indicate that all isolated fungi including

Alternaria, Aspergillus, Chaetomium, Fusarium,

Geotrichum, Mucor, Penecillium, Phytophthora,

Rhizoctonia and Trichoderma were soil borne and

prevalence of Aspergillus and Fusarium was maximum

in rhizosphere of plants from Wazirabad and Mandi

Bahudin.

Phytophthora was present in roots of tobacco only at the

site of Mandi Bahudin while Chaetomium and

Geotrichum were only present in soil of Mandi Bahudin.

Alternaria alternata, Fusarium oxysporum, Phytophthora

nicotianae and Rhizoctonia solani were pathogenic that

causes brown spot, Fusarium wilt, black shank and root

rot disease in tobacco respectively (Rodriguez, 2007,

Gveroska and Ziberoski, 2011). Aspergillus, Chaetomium,

Geotrichum, Mucor, Penicillium was non-pathogenic to

tobacco crop.

Trichoderma harzianum was also present in rhizospere

of Mandi Bahudin that also used as fungicides (Harman,

2006). Of the fungal isolates 70% belonged to phylum

Ascomycota while Basidiomycota, Oomycota and

Zygomycota contain 10% fungi isolated.

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