Indian Phytopath. ';0 (2) : 235-241 (1997)

Screening of higher plants for fungitoxicity againstRhizoctonia solani in vitro

V. KURUCHEVE, J. GERARD EZHILAN and J. JAYARAJ

Plant Pathology Department, Faculty of Agriculture, Annamalai University, Annamalainagar 608 002

ABSTRACT : Thirteen plant species were screened in vitro for the fungitoxicity against Rhizoctonia solani Kuhn,the causal agent of sheath blight of paddy. Out of these, the maximum inhibition of mycelial growth was observedin cold water extract of Prospopis juliflora followed by Thevetia peruviana. On hot water extraction, T peruvianastood first followed by P. juliflora extract. The complete inhibition of sclerotial production w.as recorded in the coldwater extracts of Caesalpinia pulcherrima, Eucalyptus globulus and Lawsonia inermis and in the hot water extractsof Calotropis gigantea, Ocimum sanctum and P. juliflora in all the concentrations tested. A similar effect was noticedwith cold and hot water extract of Pongamia glabra. The fungitoxicity of C. pulcherrima and E. globulus was notlost even after air drying and autoclaving. All the plant extracts tested withstood their fungitoxicity to a thermaltreatment at 70°C for 10 minutes. '

Keywords : Plant extracts, Rhizoctonia solani, in vitro growth

The presence of antifungal compounds inhigher plants has long been recognised as an im-portant factor to disease resistance (Mahadevan,1982). Such compounds, being biodegradable andselective in their toxicity, are considered valuablefor controlling some plant diseases (Singh andDwivedi, 1987). In the present study, thirteen plantspecies belonging to different families werescreened for the funigitoxicity against R. solani.

MATERIALS AND METHODS

Fresh leaf samples of Allium cepa, Azadirachtaindica, Caesalpinia pulcherrima, Eucalyptusgobulus, Calotropis gigantea, Ipomoea carnia,Lawsonia inermis, Ocimum sanctum, Partheniumhysterophorus, Piper betel, Pongamia glabra,Prosopisjuliflora and Thevitia peruviana were

Received for publication September 19, 1995.

collected, and cold and hot water extracts wereprepared (Gerard Ezhilan et aI., 1994). For coldwater extraction, fresh plant leaves were washedwith tap water and sterile water. If was then pro-cessed with sterile distilled water at 1 ml/g oftissue (1: 1 V/W) with a mixie and filtered througha doubled layered cheese cloth. This formed thestandard plant extract solution (100 per cent).

For hot water extraction, the plant materialswere chopped and plunged in required quantity ofwater (1: 1 WIV) taken in a beaker and heated overa waterbath at 80°C for 10 minutes. The materialswere then processed with a mixie and strainedthrough cheese cloth. It gave the standard plantextract solution (100 per cent). The extracts weresubjected to low speed centrifugation (5000 rpm for5 min) and the clear supernatants were diluted withsterile distilled water to arrive at the required con-centrations (2.5, 5 and 10 per cent).

236 Indian Phytopathology

III vitro evaluation of antifungal effect ofplant products on the radial growth andsclerotia I production of R. solani

Czapek's agar medium was prepared in 250mlErlenmeyer flasks. Aqueous plant extracts of 1.25,2.5 and 5 ml were added to 48.75, 47.50 and 45.00ml aliquots respectively in the flasks so as to get thefinal concentrations of 2.5, 5 and 10 per cent of theextracts in the medium. The medium was pouredinto petriplates at 15 ml/plate and, after solidifica-tion, they were inoculated with 12mm fungal discsand incubated (28°C). Four replicates were main-tained. Medium with sterile distilled water served ascontrol and bavistin (500ppm) served as fungicidecontrol. The diameter of the mycelial growth wasmeasured on the 7th day and the sclerotial counttaken on 15th day of incubation.

Evaluation of physical factors on thefungitoxicity of plant products

Air drying

Fresh leaves were allowed to air dry for 20days at room temperature. The air-dried leaveswere powdered and the aqueous extracts wereprepared as earlier.

Autoclaving

Conical flasks containing the Czapek's agarmedium incorporated with 10 per cent (v/v) ofaqueous plant extracts (10%) were autoclaved at15 Ibs pressure for 20 minutes.

Tnboth the air drying and autoclaving experi-ments, Czapek's agar medium containing 10 percent aqueous extracts were inoculated with thefungal disc and the radial growth was recorded.

Temperature and storage

Leaf extracts (10 per cent) were taken in ster-ilized conical flasks and stored at room tempera-ture (28°C) for 9 days. The extracts were alsoexposed to varying temperatures, viz., 50, 60, 70°Cfor 10 minutes. The antifungal activity was testedat regular intervals by observing the mycelialgrowth. In all these experiments, bavistin (500ppm) and sterile distilled water served as controls.

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RESULTS AND DISCUSSION

Mycelial growth and sclerotia I production

The fungitoxicity of plant products againstsheath blight pathogen varied with concentration.In cold water extracts, the maximum inhibition offungal growth was observed in p. juliflora (22.5mm)followed by T. peruviana (34.0mm). But, in hotwater extracts, T. peruviana was more effectivethan P. juliflora (Table 1). In both the cases, thedegree of inhibition increased along with concen-trations of extracts. Similar antifungal effect hasbeen noticed in several plant species. Root extractsof P. juliflora was found to inhibit the germinationof Dreschlera spores completely (Charya et al.,1979). P. glabra leaf extract was found to inhibitthe mycelial growth of R. solani by 45 per cent(Mishra and Tewari, 1990). Cold water extracts ofL. inermis, 0. sanctum and P. hysterophorus showedabout 40-45 per cent inhibition of the test fungi.fungitoxicity of L. inermis and O. sanctum on dif-ferent pathogens were reported by several workers(Charya et al., 1979; Asthana et al., 1986). Naiduand John (1981) reported that the leaf extract of P.hysterophorus inhibited the growth of R. solaniand Sclerotium ro/fsii.

Cold water extracts of C. pulcherrima, E.globulus and L. inermis, and hot water extracts ofC. gigantea, 0. sanctum and P. juliflora com-pletely inhibited the sclerotial production in allthe concentrations tested. P. glabra leaf extractalso showed a similar effect irrespective of themethod of extraction. In higher concentrations, C.gigantea, 0. sanctum and P. juliflora (cold waterextraction), and C. pulcherrima and P.hysterophorus (hot water extraction) extracts com-pletely inhibited the sclerotial production of R.solani. T. peruviana extract also exhibited a simi-lar effect at higher concentrations (Table 2).

The inhibitory effect of the plant products onthe sclerotial production might be attributed to thepresence of some antifungal ingredients. Singhand Dwivedi (1987) observed the fungitoxic' activ-ity of the oils of E. globulus against the sclerotialproduction of Sclerotium ro/fsii.

Table 1. Effect of cold water extracts of different plant species on the linear growth" and sclerotial production+e of R. so/ani '<aTreatment Source Linear growth , Sclerotial production Vl

0,-...No. 2.5% 5% 10% 2.5% 5% 10% N

"'--'

TI Allium cepa 90.0 90.0 90.0 57.0 23.0 13.0 \0\0

(0.0) (0.0) (0.0) (-40.6) (-76.0) (-86.4) .::l

T2 Azadirachta indica 90.0 90.0 90.0 33.0 102.0 106.3(0.0) (0.0) (0.0) (-65.6) (+6.2) (+10.7)

T3 Caesalpinia pulcherrima 50.5 43.2 41.7 0.0 0.0 0.0(-43.8) (-52.0) (-53.6) (-100.0) (-100.0) (-100.0)

T4 Calotropis gigantea 67.8 55.2 52.8 23.0 0.0 0.0(-24.6) (-38.6) (-41.3) (-76.0) (-100.0) (-100.0)

T5 Eucalyptus globulus 43.2 41.2 37.2 0.0 0.0 0.0(-52.0) (-54.2) (-58.6) (-100.0) (-100.0) (-100.0)

T6 Ipomea carnea 87.7 88.3 88.7 66.6 77.0 126.7(-2.5) (-1.8) (-1.4) (-31.2) (-19.7) (+31.9)

T7 Lawsonia inermis 51.3 49.0 46.0 0.0 0.0 0.0(-43.0) (-45.5) (-48.8) (-100.0) (-100.0) (-100.0)

T8 Ocimum sanctum 78.0 64.8 45.2 53.0 46.0 0.0(-13.3) (-28.0) (-49.7) (-44.7) (-52.0) (-100.0)

T9 Parthenium hysterophorus 64.7 59.8 43.5 13.6 17.0 29.0(-28.1) (-33.5) (-51.6) (-86.5) (-82.2) (-69.7)

TIO Piper betel 89.0 89.7 65.2 118.0 117.0 35.6(-1.1) (-0.3) (-27.5) (+22.9) (+21.8) (-62.8)

TII Pongamia glabra 50.0 46.3 45.8 0.0 0.0 0.0(-44.4) (-48.5) (-49.1) (-100.0) (-100.0) (-100.0)

TI2 Prosopis juliflora 74.2 60.1 22.5 43.0 13.7 0.0~(-17.5) (-33.2) (-75.0) (-55.2) (-86.6) (-100.0) e.:

T13 Thevitia peruviana 63.2 53.7 34.0 43.0 23.0 0.0 I:l:s(-29.7) (40.3) (-62.2) (-55.2) (-76.0) (-100.0) "tI

TI4 Bavistin 50 WP (500 ppm) 12.0 0.0 ~(-86.67) (-100.0) .g

TI5 Control 90.0 96.0 I:l

~S.E. 0.29 0.05 0-CD (0.05) 0.58 0.10 ~

• - Mean diameter of the mycelial growth (mm) .•• - Mean sclerotial numberlPetri plate.

NFigures in the parantheses are per cent increase (+) or decrease (-) over control w

-.I

Table 2. Effect of hot water extracts of different plant species on linear growth" and sclerotial production+" of R. solani tvw00

Treatment Source Linear growth Sclerotial production

No. 2.5% 5% 10% 2.5% 5% 10% ~~I:l

TI Allium cepa 90.0 90.0 90.0 54.3 79.0 121.0 ::s

(0.0) (0.0) (0.0) (-43.4) (-17.7) (+26.0) "i:l~T2 Azadirachta indica 90.0 90.0 90.0 84.3 54.3 35.0 ..•

(0.0) (0.0) (0.0) (-12.1) (-43.4) (-63.5).gI:l

T3 Caesalpinia pulcherrima 61.5 42.7 34.8 54.0 0.0 0.0 s(-37.6) (-52.5) (-61.3) (-43.7) (-100.0) (-100.0)

c

~T4 Calotropis gigantea 68.3 57.2 50.8 0.0 0.0 0.0(-24.1) (-36.4) (-43.5) (-100.0) (-100.0) (-100.0)

T5 Eucalytpus globulus 70.4 56.0 41.7 69.0 53.3 5.6(-21.7) (-37.7) (-53.6) (-28.1) (-44.4) (-94.0)

T6 Ipomoea carnea 89.7 87.3 88.3 78.0 61.0 53.3(-0.3) (-3.0) (-1. 8) (-78.1) (-69.4) (-44.4)

T7 Lawsonia inermis 60.0 55.8 51.8 21.9 29.3 42.0(-33.3) (-38.0) (-40.2) (-78.1) (-69.4) (-56.2)

T8 Ocimum sanctum 48.4 42.3 41.5 0.0 0.0 0.0(-46.2) (-53.0) (-53.8) (-100.0) (-100.0) (-100.0)

T9 Parthenium hysterophorus 76.3 45.8 41.7 39.0 0.0 0.0(-15.2) (-49.1) (-53.7) (-59.3) (-100.0) (-100.0)

TlO Piper betel 89.3 89.0 71.0 81.0 70.3 7.6(-0.78) (-1.1) (-21.1 ) (-15.6) (-26.7) (-92.6)

Tli Pongamia glabra 49.3 47.2 44.7 0.0 0.0 0.0(-45.2) (-47.5) (-50.3) (-100.0) (-100.0) (-100.0)

Tl2 Prosopis juliflora 22.8 20.1 20.5 0.0 0.0 0.0(-74.6) (-77.6) (-77.2) (-100.0) (-100.0) (-100.0)

TI3 Thevitia peruviana 52.5 40.5 13.3 67.0 0.0 0.0(-41.6) (-55.0) (-85.2) (-30.2) (-100.0) (-100.0)

Tl4 Bavistin 50 WP (500 ppm) 12.0 0.0(-86.6) (-100.0)

Tl5 Control 90.0 96.0 '<S.E. 0.86 0.05 ~CD (0.05) 1.71 0.09 VI

0,-.,• - Mean diameter of the mycelia! growth (mm). tv

'-'•• - Mean sclerotia! numberlPetri plate. -\0Figures in the parantheses are per cent increase (+) or decrease (-) over control. \0

~

'<2-Vl0,-.,tv'-'-\0\0

Table 3. Effect of certain physical factors on the fungitoxicity* of plant products ~

Trt. Source Cold Air Auto- Temperature Storage (days)No. water drying c1aving

extract 50°C 60°C 70°C 3 6 9

Tl Caesalpinia 41.7 41.7 41.0 40.5 41.0 42.2 45.5 46.0 47.5 49.0pulcherrima (-56.6) (-53.7) (-54.4) (-55.0) (-54.4) (-53.1) (-49.4) (-48.4) (-47.2) (-45.3)

T2 Eucalyptus globulus 37.2 36.7 36.0 34.3 34.5 35.0 62.5 63.5 64.5 65.5(-58.6) (-59.2) (-60.0) (-61.8) (-61.6) (-61.1 ) (-30.5) (-29.4) (-28.3) (-27.2)

T3 Parthenium 43.5 44.8 90.0 44.8 45.6 47.2 51.7 59.8 72.0 82.8hysterophorus (-51.6) (-50.2) (0.0) (-50.2) (-49.3) (-47.5) (-42.5) (-33.5) (-20.0) (-8.0)

T4 Pongamia glabra 45.8 61.0 90.0 46.4 47.7 47.7 55.5 61.0 74.2 87.5(-49.1) (-32.2) (0.0) (-48.4) (-47.0) (-47.0) (-38.3) (-32.2) (-17.5) (-2.7)

T5 Prosopis juliflora 20.0 19.8 42.2 18.5 22.0 23.5 22.5 28.0 49.8 82.0(-77. 7) (-78.0) (-53.1) (-79.4) (-75.5) (-73.8) (-75.0) (-68.8) (-44.6) (-8.8)

T6 Thevitia peruviana 34.0 45.5 33.3 30.7 33.5 35.0 42.3 53.7 69.8 84.5(-62.2) (-49.4) (-63.0) (-65.8) (-62.7) (-61.1 ) (-53.0) (-40.3) (-22.4) (-6.1)

T7 Bavistin 12.0 12.0 12.0 12.0 12.0 12.0 12.0 12.0 12.0 12.0~(-86.6) (-86.6) (-86.6) (-86.6) (-86.6) (-86.6) (-86.6) (-86.6) (-86.6) (-86.6) E.,.t:l

T8 Control 90.0 90.0 90.0 90.0 90.0 90.0 90.0 90.0 90.0 90.0 :s'"tl

*- Mean diameter of the mycelial growth (mm) ~-.Figures in parentheses are per cent increase (+) or decrease (-) over control.

.gt:lS-o-~

tvW\0

240 Indian Phytopathology

Among the plant products screened, A. cepa, A.indica and I carnea had no inhibitory effect on themycelial growth of R solani. However, Dubey andDwivedi (1991) found that onion leaf extract at 5per cent concentration checked the growth ofMacrophomina phaseolina by 39 per cent. PremlathaDath (1982) reported that A. indica and I carneawere ineffective against R. solani in vivo. However,Lakshrnanan et al. (1990) reported that neem leafextract was effective against Thanetophoruscucumeris, the causal agent of collar rot of Phaseolusaureus. At the same time, onion in hot water extrac-tion, and neem and I carnea in cold water extrac-tion •stimulated the sclerotial production at higherconcentrations. The stimulatory effect of plant prod-ucts might be due to the presence of some stimula-tory constituents that might favour the formation ofsclerotia. Some extracts like onion in cold water andPiper betel in hot water extraction at 10 per centfavoured the mycelial growth of the test fungus butthe sclerotial production was reduced by 86.5 percent and 92 per cent over control, respectively. Thismight be due to the slow absorption of thefungitoxicants present in the extracts incorporatedin the media as well as inhibition of mycelial aggre-gation for the sclerotial formation. The variation inthe inhibitory effect of various plant extracts may beattributed to the quantitative and qualitative differ-ences in the antifungal principles.

Effect of physical factors on the fungitoxicityof plant products

Air drying

Air-dried leaves of C. pulcherrima, E.globulus, P. hysterophorus and P. juliflora re-tained both mycelial and sclerotial inhibitory ef-fect. Other sources had lost the effect on air dry-ing (Table 3). The loss of fungitoxicity on airdrying might be due to the loss of certain antifun-gal principles. Air-dried plant materials had par-tial toxicity as compared to fresh leaves (GerardEzhilan et al., 1994).

Autoclaving

The extracts of C. pulcherrima, E. globulus

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and T. peruviana retained both mycelial and scle-rotial inhibitory effect even after autoclaving. Thismight be due to the thermostable nature of activeprinciples present in these extracts. Several work-ers reported that the plant extracts retained theinhibitory effect even after autoclaving (Asthanaet al., 1986). However, the other extracts lost theirinhibitory effect after autoclaving.

Temperature

All the extracts retained the mycelial inhibi-tory effect when exposed to 70°C but not afterautoclaving. Arnold (1958) reported that thefungitoxicity of different plant extracts were ther-mostable upto 120°C. This is in line with thepresent findings.

Storage

In the case of C. pulcherrima, there was notmuch reduction in the inhibitory effect even after9 days of storage. In all the leaf extracts, therewas gradual reduction in the inhibitory effect alongwith time. Lapis and Dumancas (I978) reportedthat the fungitoxic activity of plant extracts de-creased with time and it might be due to transfor-mation of the active compound to inactive formby reaction with atmosphere and light.

ACKNOWLEDGEMENT

The authors are thankful to Dr. R.Ramabadran Professor and Head, Plant PathologyDepartment, Annamalai University,Annamalaingar for providing necessary facilities.

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