characterization and in vitro … and in vitro management of rhizoctonia solani on cabbage and...

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Contemporary Research in India (ISSN 2231-2137): Vol. 7: Issue: 3 September, 2017

CHARACTERIZATION AND IN VITRO MANAGEMENT OF

RHIZOCTONIA SOLANI ON CABBAGE AND

CAULIFLOWER IN KERALA

Nusrath Beegum C.H1, Yamini Varma, C.K 2, Anita Cherian. K 3,

Reshmy Viyararaghavan4, Rashmi, C.R5, Mohamed Anees6 1Msc. Scholar, 2Associate Professor, 5, 6Teaching Assistants, Dept. of Plant Pathology,

College of Agriculture, Padannakkad, Kasargod 3Professor and Head, 4Assistant Professor, Dept. of Plant Pathology, College of Horticulture,

Vellanikkara, Thrissur

Received: 14/08/2017 Edited: 21/08/2017 Accepted: 28/08/2017

Abstract: Characterization of the fungus causing leaf blight in cabbage and cauliflower, Rhizoctonia solani was done using

morphological and cultural characters and was confirmed by molecular methods. The purposive sampling survey conducted at four

districts of Kerala showed a maximum disease severity of Rhizoctonia leaf blight (68.3 %) from Chullikkara area of Kasaragod,

Kerala. Initial symptom of foliar blight was appearance of small, irregular bluish green lesions and later changing to light brown

in colour. Many lesions coalesced to form large patches causing blighting and drying in later stage. In severe cases, the diseased

portion became papery and got withered away. At this stage yellowing around the blighted portion was also observed. Infection

produced in the unopened leaves caused head rot in cabbage. Under lower humidity, leaves dried up defoliation occurred. The

result of in vitro evaluation of the fungicides revealed that tebuconzole 5EC (Folicure), carbendazim, copper oxychloride 50 WP

(Blitox), trifloxystrobin 25% + tebuconazole 50% (Nativo) and propineb 70 WP (Antracol) at all the three concentrations

showed cent per cent inhibition of the pathogen. Bordeaux mixture also showed 100% inhibition at 1 and 1.5 %.

Keywords: Cabbage, cauliflower, Rhizoctonia solani.

Introduction

Cabbage and cauliflower are the most

economically important cole crops of the family

Brassicaceae. India is the second largest producer of

cabbage next to China, accounting for 16.55 per cent

of the world area. In Kerala, cabbage and cauliflower

are grown as seasonal crop during August to

February months. Presently, availability of many

tropical varieties of cabbage and cauliflower suited to

Kerala has led to an increase in the production of

these crops to a commercial level in many districts.

However promising cultivars of cabbage and

cauliflower are under a great threat for profitable

cultivation due to the attack of several biotic factors,

among which disease causing pathogens are the most

destructive ones. The warm humid tropical climatic

conditions of Kerala attract many fungal pathogens

especially in the intensively cultivated tracts. Leaf

blight caused by Rhizoctonia is upcoming as major

disease in cabbage and cauliflower in Kerala and so

far there are no detailed studies undertaken on this

disease. In this context, surveys were conducted in

different selected districts of Kerala and preliminary

studies were made on the management of the

pathogen under in vitro conditions.

Material and methods

Isolation of fungi

Purposive sampling surveys were conducted

in nine locations of four districts viz., Kasargod,

Thrissur, Wayanad and Idukki for the collection of

diseased samples of cabbage and cauliflower and

recorded the disease incidence and severity during

crop season of 2015-17. A total of 11 samples of leaf

blight caused by Rhizoctonia were collected and

examined. The samples were washed under running

tap water and cut into small bits consisting of both

healthy and infected portions using a sterile blade

and were disinfected with sodium hypochlorite (1%)

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for one minute. After three washings using sterilized

distilled water, the samples were placed on solidified

Potato Dextrose Agar (PDA) medium aseptically in

sterile Petri dishes. After incubation at room

temperature (28± 2oC), the fungal growth from

second to sixth days of incubation was subsequently

sub cultured to solidified PDA in sterile Petri dishes.

For use in further studies the fungi were purified by

single spore isolation and single hyphal tip method

and the purified pathogen cultures transferred in to

PDA slants.

Cultural and Morphological characterization

The cultural characters were studied by

recording the visual observations on the growth of

the pathogens.The culture discs of the pathogen on

PDA with a diameter of 5mm was placed at the

centre of PDA plates and incubated at 28±2oC.

Morphological characters of the fungus were studied

based on observations under light microscope.

Mycelia from the PDA scooped out and mounted in

Lactophenol cotton blue. A research microscope

(Carl zeiss AXIO A1) was used for observations and

ZEN imaging software were used for making photo

micrographs. Identification up to species level was

carried out at Rajiv Gandhi Centre for Biotechnology

(RGCB), Thiruvananthapuram by ITS sequencing.

Pathogenicity test

Fresh, healthy non infected plant parts of

cabbage and cauliflower were collected, brought to

the laboratory and washed under running water

followed by surface sterilization using 70% ethanol.

Artificial inoculation of pathogen carried out prior to

which the concerned plant parts injured using sterile

needle followed by the placing of the fungal

mycelium at the site of injury. From the seven day

old culture of test fungus mycelial discs of size 8 mm

diameter taken and inoculated in inverted position

on detached leaves. The site of inoculation covered

with moist cotton. After inoculation, leaves were

kept under high humidity and incubated at room

temperature till the symptoms appeared. The healthy

plant parts with injury but without inoculation with

the fungal mycelium served as control.

Invitro evaluation of chemical fungicides

against the pathogen

The experiment was conducted as CRD with

three replications. Invitro evaluation of the fungicides

was done by poisoned food technique. Ten

commercially available fungicides were used for the

study. The fungicides were mancozeb, propineb,

trifloxystrobin + tebuconazole, copper hydroxide,

copper oxychloride, Bordeaux mixture,

chlorothalonil, carbendazim, azoxystrobin and

tebuconazole. Three concentrations lower,

recommended and higher concentrations were used

for the study. The desired concentration of the

fungicide weighed out and added to the 50ml sterile

distilled water. This was then added to the 50 ml

molten double strength PDA. About 15-20 ml of the

amended medium poured to the sterile petri plates

under aseptic condition. Using a disc cutter 8 mm

diameter mycelial disc scooped out and placed at the

centre of the plate. The pates without fungicides

served as control. The percentage inhibition was

calculated using the formula

I = C-T / C × 100

Where,

I – Percentage inhibition

C - Growth of pathogen in control plate

T - Growth of pathogen in treatment plate

Statistical analysis

All the data were analysed using OPSTAT software.

Results

Isolation of fungi

The results of the survey conducted at four

districts of Kerala showed a maximum disease

severity of Rhizoctonia leaf blight (68. 3 per cent)

from Chullikkara area of Kasaragod, Kerala. The

disease was found to be more severe in cabbage

compared to cauliflower. Initial symptom of foliar

blight of cabbage and cauliflower was appearance of

small, irregular bluish green lesions and later

changing to light brown in colour. Many lesions

coalesced to form large patches causing blighting and

drying in later stage. Generally older leaves are first

affected. In severe cases, the diseased portion

became papery and got withered away. At this stage

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yellowing around the blighted portion was also

observed. Sometimes infection appeared as wet

decay of base of outer leaves. Infection produced in

the unopened leaves caused head rot in cabbage. On

the heads bluish green lesions developed, which

enlarged in size. Wet rot was seen which extended to

deeper layers of head causing complete rotting.

Under lower humidity, leaves dried up defoliation

occurred. If humidity is more, infected leaves became

black in colour due to severe rotting. Creamy white

to light brown mycelia could be observed on the

affected foliage. Often such mycelia aggregated to

form hard globular irregular sclerotial bodies which

were initially white turning to brown with a size of 1-

4 mm in diameter. In rare cases petioles of leaves

also showed sunken lesions.

Cultural and Morphological characterisation

The colony initially appeared as creamy white

in colour later turning into light brown with

abundant fluffy aerial mycelium. The underside of

the plate appeared dark brown in the centre and light

brown in the periphery (Plate 1). The pathogen

produced light brown to coffee brown, globular to

irregular shaped 1-4mm diameter sclerotial bodies on

the medium 7 days after inoculation. The mycelia of

the pathogen showed right angled branching (Plate 2

a) and a characteristic septum at the point of origin

of the right angle. The hyphae showed a

characteristic constriction at the branching point

(Plate 2b). Hyphal anastomosis was also observed

(Plate 3). The thickness of hyphae ranged from 5.46-

8.24µm. The hyphal cells were barrel shaped and

light brown in colour. The characters showedby the

pathogen were in accordance with the reports by

Parmeter et al. (1970). Based on these cultural and

morphological characters, the fungal pathogen was

confirmed as Rhizoctonia sp.

Molecular analysis

Comparison of nucleotide sequence of

Rhizoctonia culture revealed that it showed 100 per

cent identity with Rhizoctoniasolani strain HPSnG

(AccessionKF959672.1) and R. solani strain AG 1-IA

(AccessionJX089962.1). With R. solani AG-1 IA

isolate CSU8 (AccessionKX674527.1), R. solani AG-1

IA isolate CSU4 (AccessionKX674526.1),R.solani

AG-1 IA isolate CSU1 (AccessionKX674525.1) it

showed 99 per cent identity. Hence sequence analysis

of the Rhizoctonia culture showed homology with R.

solani having 100 per cent identity and cent per cent

query coverage as the same was identified earlier

through cultural and morphological characterisation

Pathogenicity test

Initial symptoms were produced within two

to three days of inoculation. Small water soaked dark

green lesions were developed initially on the lamina

which later produced blighting of whole area. Koch’s

postulates were fulfilled by re-isolating the pathogens

from the lesions present on leaves. Control plants

remained symptomless.

In vitro evaluation of fungicides against the

pathogens

The result of in vitro evaluation of the

fungicides against againstR. solani revealed that at the

recommended concentration, propineb,

trifloxystrobin + tebuconazole, copper oxychloride,

carbendazim, tebuconazole, Bordeaux mixture were

the effective fungicide with complete inhibition of

the pathogen. However copper hydroxide gave more

than 96 per cent inhibition of R.solani. Azoxystrobin

was the least effective fungicide with only 39.24 per

cent inhibition.

Discussion

In this study, the fungus that causes leaf

blight in cabbage and cauliflower were characterized

using morphological and cultural characters and the

pathogen Rhizoctonia solani was confirmed by

molecular methods. The pathogen R. solani is

reported to cause blights, root rots, and web blight

diseases in many crops like vegetable cowpea,

amaranthus, rice, etc. (Lakshmanan et al., 1979;

Gokulapalan et al., 2000, SanthaKumari and

RehumathNiza, 2005). Rashmi et al. (2016) reported

leaf blight of cabbage and cauliflower by R. solani

from Kerala and found the disease to be causing 5 to

20 % losses at Kasaragod district of Kerala. Kamala

Nayar et al. (1996) and Gokulapalan et al. (2000)

reported foliar blight of Amaranthus to be a major

problem in cultivation this green leafy vegetable. R.

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Solani causes banded blight and sheathblight disease

in rice (SanthaKumari and RehumathNiza, 2005).

Abawi and Martin, (1985) reported a similar

foliarblight disease in cabbage caused by R. solani in

New York State and observed that sclerotia and

mycelial fragments of R solani free in the soil or

associated with the organic debris will act as the

source of inoculum for leaf blight of cabbage.

The results of the in vitro efficacy of

fungicides revealed propineb, trifloxystrobin +

tebuconazole, copper oxychloride, carbendazim,

tebuconazole, Bordeaux mixture to be effective,

giving complete inhibition of the pathogen at the

field concentration. Similar results were obtained for

Sriraj et al. (2014) where, even at the lowest

concentration of 10ppm, trifloxystrobin +

tebuconazole and carbendazim recorded

effectiveness against Rhizoctonia.

Future line of research can be focused on the

management measures to be taken at field level.

References

Abawi GS, Martin SB. (1985). Rhizoctonia foliar blight ofcabbage in New York State.Plant Dis.; 69:158-161.

Gokulapalan C, Kamala Nayar, Umamaheshwaran K.2000. Foliar blight of Amaranthus caused by Rhizoctonia

solani Kuhn.J Mycol. Pl. Pathol. 30:139-241.

Kamala Nayar, Gokulapalan C, Nair MC. 1996.A new foliarblight of Amaranthus caused by Rhizoctonia

solani.Indian Phytopath.49:407.

Lakshmanan P, Nair MC, Menon MR. (1979). Collar rot and webblight of cowpea caused by Rhizoctonia solani

in Kerala, India. Pl. Dis. Reporter 63:410-413.

Parmeter JR, Jr. Whitney HS, Platt WD. 1970. Affinities of some Rhizoctonia species that resemble mycelium

of Thanetephoruscucumeris.Phytopatholog 57:218-223.

Rashmi, C.R, Mohamed Anees, Yamini Varma, C.Kand Govindan, M.2016. First report on foliar blight caused

by Rhizoctonia solani on cabbage (Brassica oleracea var capitata) and cauliflower (Brassica oleracea var botrytis)

from India. International Journal of Multidisciplinary Research and Development .3(2):6-8

SanthaKumari P, RehumathNiza TJ. 2005. Propiconazole- ANew Fungicide for Sheath Blight of Paddy.

Karnataka J. Agric. Sci. 18(3):833-835.

Sriraj, P.P, Sundravadana,S, Adhipathi and Alice, D.2014. Efficacy of fungicides, botanicals and bioagentsn

against Rhizoctonia solani inciting leaf blght on turmeric. African Journal of Microbiology Research,

8(36):3284-3294

Table 1: Efficacy of fungicides on inhibition of mycelial growth of Rhizoctonia solani

Treatments

Chemical Fungicides (concentrations)

Trade name

Inhibition (%)* C-1 C-2 C-3

T1 Mancozeb ( 0.2, 0.3,0.4 )

Mega M-45 86.67 (68.60)

87.78 (69.54)

94.81 (76.93)

T2 Copper hydroxide ( 0.1,0.2,0.3 )

Kocide 55.19 (47.96)

96.30 (83.50)

100.00 (90.000)

T3 Propineb (0.2,0.3,0.4)

Anthracol 100.00 (90.00)

100.00 (90.00)

100.00 (90.00)

T4 Trifloxystrobin + Tebuconazole (0.02, 0.03,0.04)

Nativo

100.00 (90.00)

100.00 (90.00)

100.00 (90.00)

T5 Copper oxychloride (0.1, 0.2,0.3)

Blitox 100.00 (90.00)

100.00 (90.00)

100.00 (90.00)

T6 Chlorothalonil (0.05, 0.1, 0.15 )

Kavach 91.48 (73.10)

93.33 (75.01)

93.33 (75.00)

T7 Carbendazim (0.05, 0.1, 0.15 )

Megastin 100.00 (90.00)

100.00 (90.00)

100.00 (90.00)

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T8 Azoxystrobin (0.05, 0.1, 0.15 )

Amistar

12.22 (20.44)

39.24 (38.72)

42.59 (40.67)

T9 Tebuconazole (0.05, 0.1, 0.15)

Folicur 100.00 (90.00)

100.00 (90.00)

100.00 (90.00)

T10 Bordeaux Mixture (0.5, 1.0, 1.5)

77.40 (61.60)

100.00 (90.00)

100.00 (90.00)

CD 1.803 6.84 2.967

Plate-1 Rhizoctonia solani culture on Petri dish(Front and reverse view)

Plate 2 a and b -Right angled branching of hypha

Plate 3- Hyphal anastomosis

characterization and in vitro … and in vitro management of rhizoctonia solani on cabbage and...

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