Global Advanced Research Journal of Microbiology (ISSN: 2315-5116) Vol. 2(5) pp. 089-098, May, 2013 Available online http://garj.org/garjm/index.htm Copyright © 2013 Global Advanced Research Journals

Full Length Research Paper

Effect of Neem (Azadirachta indica) leaves and seeds extract on the growth of six of the plant disease causing

fungi

Al-Hazmi, R. H. M.

Department of Biological Sciences, Faculty of Science, King Abdulaziz University, P. O. Box: 80203 Jeddah 21589, Saudi Arabia.

E-mail: rrrd2012@hotmail.com

Accepted 22 May, 2013

This study was conducted in the Department of Arid Land Agriculture of the Faculty of Meteorology, Environment and Arid land Agriculture, King Abdul Aziz University, to study the growth of plant pathogenic fungi under the influence of the aqueous and ethanolic extracts of the leaves and seeds of Azadirachta indica . The aqueous and ethanolic extracts were used in the concentrations , 1:1 , 1:10 , 1:100 , 1:1000 (v:v ) . Six of the pathogenic fungi were used , Pythium aphanidermatum, Alternaria alternata, Bipolaris sorokiniana, Fusarium oxysporium, Helminthosporium sp. and Thilaeviobsis sp. The results showed that the growth of the fungi P. aphanidermatum, A. alternaria and H. sp. was mostly inhibited by the ethanolic extract of the Neem seeds compared to the other extracts , while no inhibitory effect was noticed for the tree seed water extract on the growth of P. aphanidermatum , Alternaria alternaria and H. sp. The least affected fungus by these seeds and leaves extract was Thialeviopsis sp. The seeds and leaves extracts were mostly affective in growth retardation of the fungi when applied at the highest concentration ( 1:1 , v:v ) , P. aphanedermatum was retarded by 21.74% , A. alternaria by 33.2%, B. sorokinianae by 40.27%, F. oxysporum by 57.26%, H. sp. by 38.56%, T. sp. by 23.40%. And the ethanolic seed extract is considered most effective e growth retarding of pathological fungi than the seed water and leaf ethanolic extracts . It can be said that the water and ethanolic seeds and leaves extracts of A. indica inhibited the growth of the studied fungi at different percentages. Keywords: Neem- plant disease- pathological fungi.

INTRODUCTION Azadirachta indica (Neem), of the family is one of the mostly concerned tree by the scientist because it contains many natural substances in its different parts , leaves , seeds , bark , and has many biological activities against disease causing organisms, and it contains about 140 chemical compounds (Biswas et al. ,2002). Azadirachta indica contains complicated compounds known as the

Triterpenes, and the active agents in the tree reached about 40 types known as Tetranortripernoids or Liminoids (Jones et al.,1994). The leaves and seeds of Neem tree contain active material known as azadivactrin (Sankaram et al., 1987).

Research activities proved that Neem leaves and seeds have the ability to kill some disease causing fungi, viruses

090. Glo. Adv. Res. J. Microbiol. and parasites (Basak and Chakraborty 1968, Thind and Dahiya, 1977, Khan et al.,1991). Neem extract is very active against skin fungi which cause the ringworm disease (Narayan ,1995 ) , and a number of fungi that cause some skin disease and for curing of eczema and prevent multiplication of parasites and its distribution ( Singh et al.,1979).

Neem seeds are very rich in fatty acids amounting to about 50% of the seed weight , like oleic acid , stearic acid, palmitic acid ( Sidhu et al.,2004 ) , and its oil contains some basic amino acids (Skellon et al.,1962 ) .Many workers believe that the fatty acids in Neem seeds posses medical characteristics , curing skin diseases ( Mitra,1961 ) . Neem seeds and leaves water extract was found effective against the fungi Spherotheca pannosa var rosac which causes powdery mildew, and Ampellomyces quisaqualis ( Pasini et al.,1997 ). Neem leaves extract demonstrated a strong ability against the development of many disease causing fungi through its addition to the soil or by its direct application ( Locke ,1995 ); Tewari, 1991). The leaf extract of Neem reduced the growth of the fungi Curvularia lunata, and the germination of some pathogenic spores ( Bhowmick and Vardhan, 1981), and succeeded in resisting fruit rottening in Cucurbitaceae caused by the fungus Fusarrium equisitifolium and F. semitectum ( Krishna and Ojha, 1986), and also in reducing tomato rottening caused by the fungus Aspergillus flarus and A. niger ( Sinha and Saxena,1987 ) . The leaf water extract of Neem was found very effective against the fungal disease leaf rust of ground nut plants caused by fungi Puccinia arachidis and Mycosphaerella berkeleyi (Ghewande, 1989) .

Neem leaves water extract applied at the rate of (1:10) retarded the expansion of leaf spotting in barley plants ( Paul and Sharma,2002 ) . The effective part of the leaves ,fruit , seeds and bark of Neem against fungi may be due to the presence of malevial nimbidin ( Anonymous et al. 1984 ) . The alcoholic leaf extract of Neem was used against 13 fungi and Pythium aphanielermatum was the mostly affected ( 71.8% ) , and Pestalotiopsis mangiferae ( 46.5% ) then Bipolaris indica ( 20% ) ( Steinhauer,1996 ) . The Neem seed extract inhibited the mycelium growth of the fungus from 37 to 935 with concentrations from 50 to 1000 parts per million in petri dishes (Bankole, 1997). Ground nut late spotting disease was cured by spraying with Neem seed alcoholic extract (Alabi and Lorunju,2004). The researchers proved that the active ingredient in the neem seed extract is the nimbidin substance which was found effective against the growth of the following fungi, Fusarium oxysporium, Alternaria tennis, Rhizoctonia nodulosum ,Curvularia tuberculata (Suresh et al.,1994; Parveen and Alam( 1993 ) . Water and alcoholic Neem leaf and seed extract significantly reduced the diameter growth rate of the fungus Pyricularia oryzae that causes rice blast in petri dishes (Amadioha, 1999).

Neem seed extract retarded the growth of the fungus Gaeumannomyces graminis which causes root rottenning,

and the fungus Typhula inccarnata (Coventry and Allan , 2001).

The water and ethanol Neem seeds and leaves extract at the rate of 30:70 (v:v ) had significantly inhibited the growth of the fungi Rhizoctonia solani and Fusarium solani at percentages of 52.4% and37.5% respectively (Darwish and Shaker,2005).

In this study Azadirachta indica seeds and leaves extracts were used to test their ability to inhibit the growth of six fungi. MATERIALS AND METHODS Material: Fungi: Seeds: Seeds of Azadirachta indica were collected from trees growing inside the university compass. The mature healthy seeds were sorted out. Leaves: Azadirachta indica leaves were also collected from the same growing trees, and the healthy, green ones were sorted out. Equipments and tools: Incubator, refrigerator, oven, blender, glass bottles, petri dishes, ethyl alcohol were used. METHODS Seeds and leaves water extracts:

The seeds and leaves were washed throughly with distilled water , then blended inside an electric blender with deionized water at ratios of 1:2 ( 100gm seeds/200ml water) for seeds ,and 1:1 (100gm/100 ml water) for leaves .The extract was then infiltered through four layers of cotton net . The extract was then diluted into four concentrations, 1:1, 1:10, 1:100, 1:1000 (v:v). The alcoholic extract of seeds and leaves:

This was carried out according to the method of (Pai et al., 2004). The washed leaves were put in a well aerated shaded place to dry up, and also the cotyledons after being detached out from their seeds were throuphly washed with decontaminated water and put to dry out in a well aerated shaded place for 10 days . The seeds and leaves samples were crushed to powder , and from each 20gm samples were taken , added to 70 ml of ethyl alcohol and 30 ml of distilled water in decontaminated glass bottles . This mixture was put in a shaker away from light for one week, then infiltered to obtain the alcoholic seed and leaf extracts. This extract was poured in petri dishes and left under 40⁰C until the evaporation of all the alcohol, and the

Al-Hazmi 091 Table (1):Six species of fungi were used in this study as illustrated in the table

Infecting fungi Disease Host Source

Pythium aphanedermatum

Damping off cucumber Plant disease lab.Dept.of A.L.A.

Alternaria alternate Leaf spottingand stain barley "

Bipolaris sorokiniancae Black mold tomato Microbiology Lab.

Fusarium oxysporum Root wiltiing alfalfa Plant disease lab.Dept.A.L.A.

Helminthosporium sp. ---------- soil "

Thialeviopsis sp. ----------- soil "

Table(2): Analysis of variance of the effect of Neem extracts and their concentrations on the diameter growth of the fungi

Source of variation

DF

Pyth

ium

a

ph

ane

dde

rmatu

m

Alte

rna

ria a

ltern

ata

Bip

ola

ris s

oro

kin

ian

ae

Fu

sa

rium

oxysp

oru

m

He

lmin

thosp

oriu

m s

p.

Th

iale

vio

psis

sp.

Extract 2 6.26** 7.26** 1.21** 0.02 4.61** 0.57**

Concentration 4 3.80** 0.83** 1.59** 2.78** 1.54** 1.29**

Extractxconc 8 1.16** 0.17** 0.33** 0.67** 0.53** 0.21**

SD 45 0.10 0.01 0.03 0.008 0.03 0.04

**Significant differences at the level 0.01

powder remained With this powder dilutions with water were made as before. Growth of fungi: The fungi were grown in petri dishes containing uncontaminated potato dextrose agar (PDA) (200,20,15 gm/l respectively).

Five petri dishes were used four for the concentrations and one for the control, per fungus for both the seeds and leaves extract to give a total of 270 petri dishes. Exactly 0.5 ml of the seeds and leaves extracts were smeared on the surface of the agar in each petri dish and fungal samples each in the form of a disc 6 mm in diameter were placed in the center of each petri dish to grow. The petri

dishes were incubated at 27⁰C from 1 to 7 days before taking the average growth area for each fungus. Statistical analysis:

The factorial analysis design was used in a completely randamized Design with the extract as the main plot and the concentration as the subplot. RESULTS The results were discussed in the form of the mean diameter of the treated fungi at the different extracts at the different concentrations, expressed as percentages, compared to the untreated (control) fungi diameters. Table (2) illustrates the results of the statistical analysis, and table (3) shows the mean diameters of the growing

092. Glo. Adv. Res. J. Microbiol. Table (3): Percentage means of the fungal colony inhibition due To the effect of the extracts and their concentrations

Source of variation

Pyth

ium

ap

han

ed

de

rma

tum

Alte

rna

ria a

ltern

ata

Bip

ola

ris s

oro

kin

ian

ae

Fu

sa

rium

oxysp

oru

m

He

lmin

thosp

oriu

m s

p.

Th

iale

vio

psis

sp.

Extract

(S.W.E.) ----- ------ 3.54a 18.14b ----- 9.23b

(S.A.E.) 13.38c 29.36b 25.67c 16.18b 29.26b 5.11a

(L.A.E.) 10.34b 26.15b 15.79 16.17b 31.78b 7.53a

Concentration

(1:1) 21.74c 31.20c 40.27d 57.26d 38.56d 23.40c

(1:10) 6.26b 11.93b 20.00c 16.18b 28.39d 10.0b

(1:100) 4.0b 11.93b 8.85b 8.83b 18.65c 5.65b

(1:1000) 3.2oab 4.13a 4.43a 1.48a 9.33b 5.65b

control 6.58 2.18 2.26 2.04 2.36 3.72

LSD 0.21 0.10 0.10 0.12 0.18 0.40

SWE : seed water extract . SAE : seed alcoholic extract . LAE : leaf alcoholic extract.

LSD at 0.05 significance . Values with the same letter have no significant differences.

fungi expressed as percentages in the different treatments. Pythium aphanedermatum:

The different extracts and concentrations and their interactions have exerted high significant (p>0.01) effect on the growth of fungi, table (2). The table of mean percentages (table3) shows that the seed alcoholic extract has significantly caused the highest fungal growth inhibition retarding the growth by 13.38% , followed by the leaf alcoholic extract with 10.34% compared to the control , and the seed water extract gave no effect on the growth of P. aphanedermatum.

As for the different extract concentrations, the fungal growth rate expressed as the fungal colony diameter ,represented as percentage (table3) , show significant reduction in these diameters compared to the control , and that the colony growth percentages decreased gradually with increase in the extract concentrations . This indicate the inhibition of the fungal growth in all extract concentrations , with the significantly highest inhibition being in the highest concentration (1:1) giving a reduction in growth amounting to (21.74%) ,followed gradually in a decreasing manner by the concentrations 1:10 , 1:100 , 1:1000with 6.24% ,4.0% and 3.20% growth reduction respectively.

The interaction results (table 2) shows the significant effect of the extract and its concentrations on the fungal growth , and figure (1) shows the domination of the seed

water extract giving the highest growth inhibition rate at the highest concentration (1:1), seconded by the seed alcoholic extract and then the leaf alcoholic extract. No interaction effects were observed for the other concentrations. Alternaria alternata: The analysis of variance (table 2) illustrates very high significant effect (p<0.01) of the plant extract, the concentrations and their interactions on the growth of the fungus A. alternaria. And the mean inhibition percentages in the fungal growth (table 3) show that the seed and leaf alcoholic extracts have given the major retarding effect on the growth of the fungi with (29.36%) and (26.15%) , while the seed water extract has no inhibitory effect on the fungal growth . Consequently there was a correlation between the extract concentration and the percent of the fungal growth inhibition rate . As the extract concentration decreases from 1:1 to 1:1000 , the colony growth inhibitory percent decreases . The highest fungal growth inhibition (31.20%) was under the highest extract concentration (1:1), while the concentrations 1:10, 1:100, 1:1000 retarded the growth by 11.93 ,11. 93 and 4.14% respectively .

Figure (3) illustrates the effect of the seed water extract at the concentration (1:1) on fungal growth. The interaction

Al-Hazmi 093

Figure (1): Effect of Neem seed water extract at (1:1) concentration On diameter growth of P. aphanedematum.

Figure (3): Effect of Neem seed water extract at (1:1) concentration On diameter growth of A. alternaria.

094. Glo. Adv. Res. J. Microbiol.

Figure (5): Effect of Neem seed water extract at (1:1) concentration On diameter growth of B. sorokinnainae.

between the extract and concentration (figure 4) shows that seed water extract had the highest inhibitory effect on the fungal growth at concentration (1:1) , seconded by the leaf and seed alcoholic extract respectively . But with the other concentrations the seed alcoholic extract exerted the highest growth inhibitory. Bipolaris sorokinianae: There were high significant (p<0.01) effect of the different seed and leaf extracts and concentrations and their interactions on the growth of the fungus B. sorokinianae (table 2). The table of the fungal colony inhibitory percent means (table 3) shows that the seed alcoholic extract has given the highest fungal inhibition percent (25.67%) compared to the control, while the leaf alcoholic and seed water extract retarded the fugal growth at percents of 15.79

and 3.5% respectively. As regard the extract concentration effects the highest fungal growth inhibition was observed in the concentration 1:1 at a percentage of 40.27%, and this growth inhibition percent gradually decreased with decrease in the extract concentration from 1:1 to 1:1000 to 20.00%, 8.85% , 4.43% respectively .

Figure (5) illustrates the effect of the seed water extract at (1:1) on the diameter growth of the fungus B.sorokinianae.

Regarding the treatment interaction effect on the fungal growth, figure (6) shows the domination of the seed alcoholic extract, seconded by the leaf alcoholic and then the seed water extracts respectively with the concentrations 1:10, 1:100 and 1:1000 . But at the highest concentration (1:1) the seed water extract gave the highest

Al-Hazmi 095

Figure (7): Effect of Neem seed water extract at (1:1) concentration On diameter growth of F. oxysporium.

fungal growth inhibition compared to the two other concentrations. Fusarium oxysporum: There were high significant (p<0.01) effect of the different extracts and concentrations and their interactions on the growth of the fungus F. oxysporum table (2). The results of the mean percentages of the fungal community growth inhibition (table 3) shows that the three types of extracts exerted retarding effect on the fungal growth at the rate of 18.14 16.11 and 16.17% respectively , with no significant differences between them . There was a gradual decrease in the percentage of the fungal growth inhibition with reduction in extract concentration, and the concentration 1:1 gave the highest growth retardation percent 57.25% , followed in a decreasing manner by the concentrations

1:10 , 1:100 , 1:1000 suppressing the fungal colony growth by 16.18 , 8.81 and 1.48% respectively . Figure (7) shows the effect of the seed water extract at concentration 1:1 on the diameter growth of F. oxysporum. Figure (8) illustrates the extract concentration reaction effect on the growth of the fungus, with the seed water extract dominating exerting the highest growth inhibition at concentration (1:1) . There were no significant differences between the other treatments. Helminthosporium sp.: The results in table (2) show high significant (p<0.01) effects of the extracts, the concentrations and their interactions on the growth of the fungus H.sp. The percentage means of the magnitude of the fungal colony

096. Glo. Adv. Res. J. Microbiol.

Figure (9): Effect of Neem seed water extract at (1:1) concentration On diameter growth of Helminthosporium.

growth inhibition ( table 3) show no significant difference between the seed and leaf alcoholic extracts on the fungal growth, with the leaf extract exerting 29.26% and that of the leaf 31.78% inhibition ,with no inhibitory effect of the seed alcoholic extract.

There was a gradual decrease in the magnitude of the growth inhibitory effect with reduction in extract

concentrations, and the highest concentration (1:1) exerted the highest inhibitory effect on the fungal growth 38.56% , followed in a decreasing order by the concentrations 1:10 1:100,1:1000, with 28.39 ,18.65 .9.23% growth reduction respectively . Figure shows the reductive effect of seed water extract at concentration (1:1)on the growth of H.sp.

Al-Hazmi 097

Figure (11): Effect of Neem seed water extract at (1:1) concentration On diameter growth of Thialeviopsis sp.

As for the interaction effects, the seed alcoholic extract

dominated the other two extracts at the high concentrations 1:1 and 1:10 giving the highest growth inhibition percent , while at the low concentrations 1:100 and 1:1000 the domination was for the seed water extract followed respectively by the seed alcoholic and the leaf alcoholic extracts. Thialeviopsis sp. The results in table (2) indicate high significant effects (p<0.01) of the extracts, the concentration and their interaction on the growth of the fungus T. sp. The mean percentage values of fungal growth inhibition in table(3) show that all of the treatments have exerted inhibitory effects , with the seed water extract being more effective in retarding the colony growth than the seed and leaf alcoholic extract , by 9.23 % compared to 5.11 7.53% respectively .

As for the concentration effects , the highest concentration (1:1) exerted the highest inhibitory effect (23.40%) , followed by the concentration (1:10) with 10.0% , and then the concentrations (1:100) and (1:1000) each with 5.65% inhibition effect .Figure shows the effect of the seed water extract at the concentration (1:1) on the diameter growth of the fungus Thialeviopsis sp.

When considering the interaction effects (fig 12) , the seed water extract dominated giving the highest growth inhibitory effect seconded by the leaf and seed alcoholic extracts respectively at the high concentrations (1:1), (1:10) . And when the concentration decreased there were no significant differences between the treatments. DISCUSSION The seed water extract didn´t inhibit the fungal growth of Pythium aphanedermatum, Alternaria alternaria and Helminthosporium sp. The seed alcoholic extract was more

098. Glo. Adv. Res. J. Microbiol. effective in retarding fungal growth than the leaf alcoholic extract in P. aphanedermatum, A. alternaria, Bipolaris sorokinianae with no significant differences between the two extracts in the case of Fusarium oxysporium and H. sp. This in agreement with the finding of ( Suresh et al,1974 ; Parveen and Alam, 1993 ) who found that seed alcoholic extract inhibited the growth of F. oxysporium due to substance nimbidin .

The six fungi growth was highly affected and were inhibited to the highest percentage by the highest concentration (1:1) of the different seed and leaf extracts , and F. oxysporium being mostly affected (57.26%) , then B. sorokinianae with (40.27%) and H. sp. inhibited by (38.56%) . This is in agreement with the results obtained by many researchers , Amadioha, 1999 found that Neem seeds and leaf extracts have reduced the growth of the fungi Pyricularia oxyzae in rice . The impact on the fungal growth by the plant extract was successively reduced with reduction of the extract concentration in all of the six fungi , with the growth of the fungus H. sp. comparatively remaining unaffected with all of the extract concentrations . The least affected fungal growth by the Neem seed and leaf extract in this study was the fungus Thialeviopsis sp. The water extract of seeds and the alcoholic extract of seeds and leaves had significant inhibition effect and retarded the growth rate of the fungi . This is agreement of (Steinhauer, 1996 ) who tested the effect of Neem leaf alcoholic extract on the growth of 13 different fungi , and found significant reduction with Pythium aphandermatus being the mostly affected.

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