Extracellular enzymes activity determining the virulence of Rhizoctonia bataticola, causing root rot in soybean

Physiological and Molecular Plant Pathology 100 (2017) 49e56

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Extracellular enzymes activity determining the virulence of Rhizoctonia bataticola, causing root rot in soybean D.B. Gawade a, *, R.R. Perane a, A.P. Suryawanshi b, C.D. Deokar a a b

Department of Plant Pathology & Agril. Microbiology, MPKV, Rahuri 413 722, MS, India Department of Plant Pathology, College of Agriculture, Latur 413 512, MS, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 13 April 2017 Received in revised form 26 May 2017 Accepted 8 June 2017 Available online 17 June 2017

Enzymes play crucial role in successful host-pathogen interactions and degradation of the cell wall. The cellulolytic, hemicellulolytic, pectolytic and proteolytic enzymes produced by plant pathogens are capable of degrading major polymeric components of the host cell wall. A number of pathogenic fungi have been reported to produce cellulases, but relatively a small quantum of plant pathogens are able to degrade insoluble cellulose even after it has been physically or chemically modified to make it more susceptible to enzyme action. Present study revealed Rhizoctonia bataticola as major cause of soybean root rot. Result of the R. bataticola, 20 isolates collected from various regions of Maharashtra, seven isolates were moderate virulent and thirteen isolates highly virulent. These isolates showed wide variability in per cent root rot infection of soybean ranged from 48.00 to 69.33%. Highest extracellular enzyme activity was found in respect of cellulase (Rb-33), pectinase (Rb-33), lipase (Rb-10), protease (Rb32) and amylase (Rb-36). The per cent root rot infection was found to be highly significant and positively correlated with the enzyme viz., cellulase, pectinase and protease. The entire 20 test isolates exhibited production of extracellular enzymes under both plate culture and plant sample assay methods. Maximum enzyme activity profile from R. bataticola infected root tissues was found in respect of cellulolytic (Rb-1), polygalacturonase (Rb-32) and pectin methylesterase (Rb-32) enzyme. There was low enzyme activity even the isolates were more virulent and vice versa. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Glycine max Soybean R. bataticola Root rot Enzymes Isolates Virulence

1. Introduction Soybean [Glycine max (L.) Merril] is a major commodity traded in world market and currently the world's primary oilseed crop [1]. It is nutritious, easily digestible and one of the richest and cheapest source of proteins. In the list of potential sources of unconventional food proteins, soybean ranks at the top. Soybean seed contains approximately 37e41% proteins, 18e21% oil, 30e40% carbohydrates and 4e5% ash [2]. It is grown commercially in more than 35 countries, but mostly concentrated in USA, Brazil, Argentina, and China [3]. In India, oilseeds are grown in an area of about 27 million hectares, of which soybean occupies 10.18 million hectare (38% area), spread across length and breadth of the country [4]. Soybean root rot caused by Rhizoctonia bataticola (Pycnidial stage Macrophomina phaseolina (Tassi) Goid) is one of the important soil borne pathogens, causing yield losses upto 77% [5].

* Corresponding author. E-mail address: [email protected] (D.B. Gawade). http://dx.doi.org/10.1016/j.pmpp.2017.06.003 0885-5765/© 2017 Elsevier Ltd. All rights reserved.

Enzymes plays crucial role in successful host-pathogen interactions by the way of degradation of the cell wall [6,7]. The major components of plant cell walls are cellulose, hemicellulose and lignin, with cellulose being the most abundant component [8,9]. The cellulolytic, hemicellulolytic, pectolytic and proteolytic enzymes produced by plant pathogens are capable of degrading major polymeric components of the host cell wall [10,11]. In some hostpathogen interactions, these enzymes are thought to be the determinants of virulence [12] and also disrupt other systems of the host [13,14]. Apart from cell wall degrading enzymes secreted by a wide variety of saprophytic and phytopathogenic microorganisms [15], most pathogens produce more cellulolytic than pectolytic enzymes [16]. Lipases and proteases are the important enzymes in pathogenesis, which attack plasmalemma after degradation of cell wall by proteases along with pectolytic and cellulolytic enzymes [17]. Hydrolytic enzymes play an important role in pathogenicity by facilitating fungal penetration through the host cell wall [18]. Such as the fungal enzymes pectinases, facilitates intra and inter cellularly entry of plant pathogens into the host tissues and thereby blocking the conducting vessels leading the plants to wilt [19].

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Pectin degrading enzymes pectinases weakens plant cell wall and exposes other polymers to degrade by hemicellulases and cellulases. Pectinases are the first cell wall degrading enzymes secreted by pathogens and also virulence determinant factors [20]. Therefore, in the present study an attempt was made to analyze the virulence dependent enzyme production by R. bataticola causing dry root rot of soybean in India. Also to quantify extracellular enzyme production by plate assay method and enzyme profile activity. 2. Materials and methods 2.1. Isolation and identification Twenty isolates of R. bataticola were isolated from root rot (RR) infected soybean plants collected from various regions covering 19 districts viz., Ahmednagar, Solapur, Pune, Sangali, Kholapur, Beed, Parbhani, Nanded, Jalna, Aurangabad, Latur, Akola, Buldana, Nagpur, Amravati, Jalgaon, Nandurbar, Dhule and Hingoli of the state of Maharashtra, India. Pure cultures of these isolates were maintained on agar slant tubes in refrigerator for further studies. Based on morphological and microscope observations [21], the test fungus was identified as Rhizoctonia bataticola and further confirmed by pathogenicity test. 2.2. Virulence/pathogenicity test of the isolates Twenty test isolates of R. bataticola were subjected separately to pathogenicity test by sick soil method in pot culture, under screen house condition. Surface sterilized (0.1% HgCl2) seeds of soybean Cv. JS-335 were sown (@10 seeds/pot) in the earthen pots (25 cm dia) filled with steam sterilized potting mixture of soil: sand: fym (2:1:1) and made sick with R. bataticola isolates separately, watered as per requirement and kept in the screen house. Observations on latent period (days required for appearance of first symptom), typical root rot symptoms developed, pre- and post-emergence mortality etc. were recorded. The root rot infection was determined applying the rating scale of 0e5 [22] on the basis of root discoloration as 0 ¼ No discoloration, 1 ¼ Very few small lesions (black discoloration) on roots, 2 ¼ Lesions (black discoloration) on roots clear but less and new roots free from infection, 3 ¼ Lesions (black discoloration) on roots more and many new roots generally free from lesions, 4 ¼ Roots infected and completely discoloured (black) and root length severely restricted and 5 ¼ Plants completely dead. Per cent root rot infection was calculated by applying following formula.

Percent root rot infection ¼

plate assay method in germinating fungus [23]. 2.3.1. Cellulase enzyme production The method described by Leopold and Samsinakova [24] was used for cellulolytic activity. Autoclaved and cooled basal medium (NaNO3 0.2 g; KH2PO4 0.1 g; KCl 0.05 g; MgSO4 0.02 g; CaCl2 0.01 g; Yeast extract 0.05 g; Distilled H2O 100 ml) was aseptically poured (20 ml/plates) in sterilized glass petri plates (90 mm dia.). On solidification of the medium, these plates were inoculated aseptically by placing in the center a 5 mm mycelial disc obtained from 7 days old pure culture of R. bataticola test isolates, multiplied on agar plates and incubated at 28±2  C. After 4 days of incubation, the culture growth is flooded with 1% aqueous hexadecyl-trimethylammonium-bromide [25] and clear zone developed around the culture growth indicated activity of cellulase enzyme. The clear zone developed (mm) in a triplicate set of petri plates per test isolate was recorded and averaged. 2.3.2. Pectinase enzyme production The autoclaved and cooled basal medium comprising 5,00 ml mineral salt solution, 1 g yeast extract, 15 g agar, 5 g pectin and 5,00 ml distilled H2O [26] was aseptically poured (20 ml/plates) in sterilized glass petri plates (90 mm dia.). On solidification of the medium, these plates were inoculated aseptically by placing in the center a 5 mm mycelial disc obtained from 7days old culture of R. bataticola test isolate, multiplied on agar plates and incubated at 28±2  C. After 4 days of incubation the culture growth in plates was flooded with 1% aqueous solution of hexadecyl-trimethylammonium-bromide, which precipitated pectin in the medium and clear zone developed around the culture growth indicated activity of pectolytic enzyme. The clear zone developed (mm) in a triplicate set of petriplates per test isolate was recorded and averaged. 2.3.3. Protease enzyme production Casein soluble medium (dextrose 40 g, peptone 10 g, agar 15 g, yeast extract 3 g, casein 8 g, distilled H2O 1,000 ml at pH 7.0) was used [27]. Autoclaved and cooled medium was aseptically poured (20 ml/plates) in sterilized petriplates (90 mm dia.) and on solidification, these plates were inoculated aseptically by placing in the center a 5 mm mycelial disc obtained from 7 days old culture of R. bataticola test isolates, multiplied on agar plates and incubated at 28±2  C for 4 days. After 4 days of incubation, the culture growth in petriplates was flooding with 10% glacial acetic acid. Clear zone developed around culture growth indicated protease enzyme activity and clear zone developed (mm) in a triplicate set of petriplates per test isolate was recorded and averaged.

Disease grade observed  100 No: of plants examined  maximum disease rating

Based on disease reaction isolates were categorized into three groups: Low virulent ¼ below 30% infection, moderately virulent ¼ 30e60% infection, highly virulent ¼ above 60% infection. 2.3. Quantification of enzymes by plate assay Twenty test isolates of R. bataticola were subjected to in-vitro production of the enzymes viz., pectinase, cellulase, protease, lipase and amylase by adopting following standard protocols. Productions of protease, cellulase and pectinase enzymes were determined by

2.3.4. Lipolytic enzyme production The basal medium (peptone 10 g, NaCL 5 g, CaCl2. 2H2O 0.1 g, agar 20 g, distilled H2O 1000 ml adjusted to pH 6.0) was used [28] and olive oil was used as lipid substrate. Autoclaved and cooled medium was aseptically poured (20 ml/plates) in sterilized glass petriplates (90 mm dia.) and on solidification, these plates were inoculated aseptically by placing in the center a 5 mm mycelial disc obtained from 7 days old pure culture of R. bataticola test isolates multiplied on agar plates and incubated at 28±2  C for 4 days. Clear zone developed around the culture growth indicated lipolytic

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enzyme activity. The clear zone developed (mm) in a triplicate set of petriplates per test isolate was recorded and averaged.

Estimated the reducing sugars formed by DNS method using Dglucose as a standard [31,32].

2.3.5. Amylolytic enzyme production Amylase enzyme activity was assessed by inoculating the test isolates on nutrient agar with 1% starch and pH 6.0. The autoclaved and cooled medium was aseptically poured (20 ml/plates) in sterilized petriplates (90 mm dia.) and on solidification, these plates were inoculated aseptically by placing in the center a 5 mm mycelial disc obtained from 7 days old pure culture of R. bataticola, multiplied on agar plates and incubated at 28±2  C for 4 days. After incubation, the plates were treated with iodine and clear zone around the active colonies indicated amylolytic activity [29]. In all of the above plate assays, petriplates containing plain medium were maintained as untreated control.

2.4.4. Pectin methyl esterase (PME) PME enzyme activity was determined at 25  C by the continuous spectrophotometric assay [34]. A 200 mg root samples were homogenized with 15 ml cold 8.8% NaCL, using pestle and mortar centrifuged at 20,000 g for 10min, collected the supernatant, adjust its pH 7.5 with NaOH and used for enzyme assay.

2.4. Quantification of enzymes form plant samples All living organisms are able to obtain and use energy very rapidly because of the presence of biological catalysts called enzymes. Enzymes function under certain well defined conditions of pH, temperature, substrate concentration, cofactors etc. The optical conditions for each enzyme have to have carefully been standardized before measuring the enzyme activity. 2.4.1. Collection of soybean root samples From the earthen pots, root rot infected root samples of soybean were collected and one each sample represented respective 20 test isolates of R. bataticola. These samples were put separately in the sterile polyethylene bags and brought to the Biochemical Laboratory, Plant Pathology Department for further analysis. 2.4.2. Cellulolytic enzyme assay The enzymatic activity was determined colorimetrically by measuring reducing sugars released by hydrolysis of carboxymethyl cellulose as substrate [30]. A 50 mg root samples were homogenized with 100 mM Sodium acetate buffer (pH 6.0) containing 0.2% sodium dithionite (Na2S2O4) and 1% PVP (MW 44000), for one min in a blender, centrifuged the homogenate at 10,000 rpm for 20min. and stored the supernatant at 12  C. Adjusted the pH to 8.2 with NaOH and incubated overnight at 4  C with continuous stirring and again centrifuged, filtered through Whatman No.1 filter paper and dialyzed against water for 48 h. 2.4.2.1. Enzyme assay. The substrate incubated 2.0 ml 1% carboxymethyl cellulose and 1.0 ml 100 mM Sodium acetate buffer (pH 5.0) at 37  C and initiated the reaction by adding 1.0 ml crude enzyme and withdrawing 0.5 ml aliquot at 2 h intervals. Estimated the reducing sugars formed by DNS method using D-glucose as a standard [31,32]. 2.4.3. Polygalactouronase (PG) enzyme assay The enzyme is assayed as the amount of reducing sugar formed colorimetrically using polygalacturonic acid, a polysaccharide of pectin [33]. A 200 mg root samples were homogenized with 13 ml Tris-HCL buffer centrifuged at 15,000 rpm for 15min and the pellet was incubated for 60min in about 5 ml of extraction buffer, further centrifuged at 15,000 rpm for 30min and filtered through Whatman No.1 filter paper. 2.4.3.1. Enzyme assay. The substrate incubated 0.1 ml NH4Cl and 1 ml polygalacturonic acid at 37  C and initiated the reaction by adding 0.1 ml enzyme extract and incubated further for 30min. Terminated the reaction by adding 0.3 ml of 5% Trichloroacetic acid and centrifuged at 2,000 g for 30min and collected the supernatant.

2.4.4.1. Enzyme assay. In a cuvette, 2 ml pectin was mixed with 0.15 ml bramothymol blue and 0.83 ml water and incubated at 25  C in a circulating water bath. Determined initial absorbance at 620 nm (A620) against water blank. Started the reaction by adding 100 ml enzyme solution and measured the rate of decrease at 620 nm at an interval of 20, 40, 60 and 80 s and calculated the activity of enzyme. 3. Results 3.1. Virulence of isolates and root rot infection Variation in the virulence among the 20 isolates of R. bataticola was studied by their ability to infect and induce root rot disease in soybean crop, under greenhouse conditions. The test isolates of R. bataticola showed a wide variability in root rot infection, which ranged from 48.00 to 69.33%. Based on the relative root lesion intensity, test isolates were grouped into three categories; less, moderate, and highly virulent (Table 1). Seven isolates viz., Rb-10, Rb-13, Rb-15, Rb-17, Rb-21, Rb-23 and Rb-27 belonging to moderate virulent group showed 30 to 60% root rot infection, thirteen isolates viz., Rb-1, Rb-3, Rb-5, Rb-6, Rb-8, Rb-18, Rb-24, Rb-30, Rb32, Rb-33, Rb-36, Rb-38 and Rb-40 belonged to highly virulent group with root rot infection above 60%, while none of the isolate was found in less virulent group with less than 30% root rot infection. Among the test isolates, significantly highest root rot infection (Table 2) was exhibited by Rb-3 (69.33%), followed by Rb33 (68.00%) and Rb-32 (66.67%). It was moderate with Rb-17 Rb-23 (each 57.33%), followed by Rb-10 and Rb-15 (each 56.00%) and Rb21 and Rb-27 (each 52.00%). 3.2. Extracellular enzymes production (plate assay) by R. bataticola isolates 3.2.1. Cellulase production All twenty test isolates exhibited production of cellulase enzyme (Table 2), which was cellulase evidenced by clear zone produced (mm) around the fungus growth on CMC agar medium. R. bataticola isolates showed wide variability in cellulase enzyme production, which ranged from 19.00 to 71.00 mm (Table 2). However, it was highest with the isolate Rb-33 (71.00 mm), followed by Rb-36 (68.67 mm), Rb-38 (66.67 mm), Rb-20 (62.67 mm), Rb-3 and Rb21 (each 61.33 mm), whereas, it was lowest with the isolate Rb-5 (19.00 mm) and Rb-17 (21.00 mm). However, there was no cellulase production in the control plates. There was less cellulase enzyme production even though some of the isolates were highly or moderately virulent and vice versa. There was a highly significant positive correlation (0.688**) between the cellulase enzyme production and virulence of the test isolates (Table 3). About 60% isolates showed high level of cellulase enzyme production, 30% isolates produced moderate cellulase and 10% isolates produced lower amount of cellulase (Table 2). 3.2.2. Pectinase production Twenty test isolates of R. bataticola were screened for their

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Table 1 Grouping and virulence of Rhizoctonia bataticola isolates, inducing root rot of Soybean in Maharashtra. Category

No. of isolates

Mean RR infection (%)

Name of the isolates

II (Low virulent) III (Moderate virulent) IV (Highly virulent)

00 07 13

0.00 54.09 63.90

– Rb-10, Rb-13, Rb-15, Rb-17, Rb-21, Rb-23, Rb-27 Rb-1, Rb-3, Rb-5, Rb-6, Rb-8, Rb-18, Rb-24, Rb-30, Rb-32, Rb-33, Rb-36, Rb-38, Rb-40

Table 2 Root rot induction by R. bataticola isolates in Soybean and its influence on production of various extracellular enzymes. Sr. No.

Isolates

Root rot (%)**

Cellulase (mm)*

Pectinase (mm)*

Lipase (mm)*

Protease (mm)*

Amylase (mm)*

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 S.E. ± C.D. @ 5%

Rb-1 Rb-3 Rb-5 Rb-6 Rb-8 Rb-10 Rb-13 Rb-15 Rb-17 Rb-18 Rb-21 Rb-23 Rb-24 Rb-27 Rb-30 Rb-32 Rb-33 Rb-36 Rb-38 Rb-40 Control

65.33 (53.95) 69.33 (56.38) 61.33 (51.76) 64.00 (53.35) 61.33 (51.58) 56.00 (48.45) 48.00 (43.81) 56.00 (48.48) 57.33 (49.25) 60.00 (50.98) 52.00 (46.14) 57.33 (49.22) 60.00 (50.88) 52.00 (46.15) 62.67 (52.59) 66.67 (54.88) 68.00 (55.72) 65.33 (54.08) 64.00 (53.19) 62.67 (52.44) 0.00 (4.05) 3.41 9.75

51.33 61.67 19.00 53.33 49.67 51.67 55.00 47.67 21.00 46.33 61.33 45.67 36.67 40.00 62.67 57.00 71.00 68.67 66.67 53.00 0.00 4.11 11.74

52.00 69.33 58.33 55.67 65.00 60.33 71.67 64.67 42.67 63.00 71.00 51.33 68.67 45.67 71.33 75.00 75.67 62.33 67.33 56.00 0.00 4.10 11.70

42.33 71.67 41.33 74.67 65.33 82.33 76.00 52.00 40.33 70.33 46.67 48.00 61.33 40.67 62.67 68.67 60.33 58.00 74.67 55.00 0.00 3.64 10.41

55.67 64.67 59.67 78.00 47.67 75.33 64.33 73.67 54.00 75.00 70.67 74.33 61.33 48.00 66.00 85.33 70.67 81.67 83.33 71.33 0.00 3.80 10.84

23.67 62.67 25.33 65.67 67.67 65.33 62.67 62.33 27.67 64.67 71.67 66.33 57.00 28.00 62.33 71.00 71.33 75.33 74.67 60.33 0.00 4.14 11.83

*Mean of three replications. **Figures in parentheses are angular transformed values.

Table 3 Correlation between extracellular enzyme production, root rot induction by Rhizoctonia bataticola isolates and their virulence pattern. Particular

Cellulase

Pectinase

Lipase

Protease

Amylase

RR

RR

0.688**

0.812**

0.701**

0.809**

0.611**

1.000

RR ¼ root rot infection %, ** ¼ highly significant, r ¼ @ 5% ¼ 0.433, 1% ¼ 0.549.

potential to produce pectinase enzyme, by applying cup-plate method. Which revealed that all of the could produced the enzyme pectinase at variable degree. Pectinase enzyme production was indicated by clear zone produced (mm) around the fungus growth on pectin agar medium. Pectinase enzyme production by the test isolates ranged from 42.67 to 75.67 mm (Table 2). However, it was highest as evidenced by clear zone (mm) with the isolate Rb33 (75.67 mm), followed by Rb-32 (75.00 mm), Rb-13 (71.67 mm), Rb-21 (71.00 mm), Rb-30 (71.33 mm), Rb-3 (69.33 mm), Rb-24 (68.67 mm), Rb-38 (67.33 mm), Rb-8 (65.00 mm), Rb-15 (64.67 mm), Rb-18 (63.00 mm), Rb-36 (62.33 mm) and Rb-10 (60.33 mm), whereas, it was moderate with the isolate Rb-17 (42.67 mm) and Rb-27 (45.67 mm) and the control plate did not indicated any pectinase enzyme production. There was less pectinase enzyme production even though some of the isolates were highly or moderately virulent and vice versa. There was a highly significant positive correlation (0.812**) between the pectinase enzyme production and the virulence of the test isolates (Table 3). About 90% isolates showed very high level of pectinase enzyme production and rest 10% showed moderate level

of pectinase enzyme production (Table 2). 3.2.3. Lipase production All 20 test isolates of R. bataticola exhibited positive activity for production of lipase enzyme, which was evidenced by clear zone production (mm) around the fungal growth on peptone agar medium. The results (Table 2) indicated that, the test isolates showed a wide range of lipase production from 40.33 to 82.33 mm. However, it was highest (mm clear zone) with the isolate Rb-10 (82.33 mm), followed by Rb-13 (76.00 mm), Rb-6 (74.67 mm), Rb-38 (74.67 mm), Rb-3 (71.67 mm), Rb-32 (68.67 mm), Rb-8 (65.33 mm), Rb-30 (62.67 mm) and Rb-33 (60.33 mm), while it was moderate with the isolates any Rb-17 (40.33 mm), Rb-27 (40.67 mm), Rb-5 (41.33 mm) and Rb-1 (42.33 mm) and the control plate did not indicated lipase enzyme production. There was less lipase enzyme production even though some of the isolates ware highly or moderately virulent and vice versa. There was a highly significant positive correlation (0.701**) between the lipase enzyme production and virulence of the test isolates (Table 3). About 70% isolates showed very high level of lipase production and rest 30% showed moderate level of lipase production (Table 2). 3.2.4. Protease production The results (Table 2) revealed that, the 20 test isolates produced protease enzyme, which was evidenced by clear zone produced (mm) around the fungal growth on agar plates. R. bataticola isolate showed a wide variability in protease enzyme production which

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ranged from 47.67 to 85.33 mm. However, clear zone (mm) produced was highest with the isolate Rb-32 (85.33 mm), followed by Rb-38 (83.33 mm), Rb-36 (81.67 mm), Rb-6 (78.00 mm), Rb-10 (75.33 mm), Rb-18 (75.00 mm), Rb-23 (74.33 mm), Rb-15 (73.67 mm), Rb-40 (71.33 mm), Rb-21 (70.67 mm), Rb-33 (70.67 mm), Rb-30 (66.00 mm), Rb-3 (64.67 mm), Rb-13 (64.33 mm) and Rb-24 (61.33 mm), while it was moderate with the isolates Rb-8 (47.67 mm) and Rb-27 (48.00 mm) and the control plate did not indicated any protease production. There was less protease enzyme production even though some of the isolates were highly or moderately virulent and vice versa. There was a highly significant positive correlation (0.809**) between the protease enzyme production and the virulence (Table 3). About 90% isolates showed very high level of protease enzyme production and rest 10% isolates showed moderate level of protease enzyme production (Table 2). 3.2.5. Amylase production 20 isolates of R. bataticola tested showed their potential to produce amylase enzyme by cup-plate method, but of variable degree (Table 1), which was evidenced by clear zone produced (mm) around the fungal growth on agar medium enriched with starch. The amylase enzyme production by the test isolates ranged from 23.67 to 75.33 mm. However, it was highest (clear zone in mm) with the isolate Rb-36 (75.33 mm), followed by Rb-38 (74.67 mm), Rb-21 (71.67 mm), Rb-33 (71.33 mm), Rb-32 (71.00 mm), Rb-8 (67.67 mm), Rb-23 (66.33 mm), Rb-6 (65.67 mm), Rb-10 (65.33 mm), Rb-18 (64.67 mm), Rb-3 (62.67 mm), Rb-13 (62.67 mm), Rb-15 (62.33 mm), Rb-30 (62.33 mm) and Rb-40 (60.33 mm), while it was lower with isolates Rb-1 (23.67 mm), Rb-5 (25.33 mm), Rb-17 (27.67 mm) and Rb27 (28.00 mm) and the control plate did not indicated any amylase production. There was less amylase enzyme production even though some of the isolates were highly or moderately virulent and vice versa. There was a highly significant positive correlation (0.611**) between the amylase enzyme production and virulence (Table 3). About 80% isolates collected showed very high level of amylase enzyme production and rest 20% isolates showed low level of amylase production (Table 2). 3.3. Extracellular enzymes activity (plant samples) 3.3.1. Cellulase activity The results (Table 4) revealed that R. bataticola test isolates produced extracellular cellulase enzyme under sick soil assay in pot culture, which was evidenced by the analysis of root rot infected soybean plant specimens. There was a wide variability in cellulase enzyme activity of the test isolates which ranged from 264.38 to 299.39 mg of D-glucose/hr/ml extract. However, it was highest with the isolate Rb-1 (299.39 mg), followed by the isolates Rb-32 (295.04 mg) and Rb-3 (293.40 mg). The cellulase activity was minimum (142.62 mg) with healthy root tissues of soybean. There was less cellulase enzyme production even though some of the isolates were highly or moderately virulent and vice versa. There was a highly significant positive correlation (0.934**) between the cellulase enzyme activity and virulence of the test isolates (Table 5). 3.3.2. Polygalacturonase (PG) activity PG activity of the root extracts of Soybean plants infected individually with twenty test isolates of R. bataticola was assessed spectrophotometrically and the results (Table 4) revealed that the test isolates exhibited PG enzyme activity, which ranged from 317.36 to 354.92 mg of D-glucose/ml extract. However, it was highest

53

Table 4 Cellulase, Polygalacturonase (PG) and Pectin methylesterase (PME) activity profile of R. bataticola isolates grown in sick pot soil condition. Sr. No.

Isolate No.

1 Rb-1 2 Rb-3 3 Rb-5 4 Rb-6 5 Rb-8 6 Rb-10 7 Rb-13 8 Rb-15 9 Rb-17 10 Rb-18 11 Rb-21 12 Rb-23 13 Rb-24 14 Rb-27 15 Rb-30 16 Rb-32 17 Rb-33 18 Rb-36 19 Rb-38 20 Rb-40 21 Control S.E. ± C.D. @ 5%

Cellulase activity (mg of D-glucose/ hr/ml extract)*

PG activity (mg of D-glucose/ ml extract)*

PME activity (A620/min)*

299.39 293.40 264.28 283.88 288.78 285.78 273.54 287.14 279.80 289.87 292.04 277.89 280.07 272.45 285.24 295.04 290.96 279.80 288.78 275.99 142.62 0.016 0.048

326.07 346.75 317.36 341.31 329.88 346.21 344.57 322.26 323.34 335.86 322.80 342.94 326.61 335.32 325.52 354.92 349.47 340.22 341.31 349.47 165.48 0.012 0.036

0.204 0.272 0.171 0.158 0.176 0.167 0.161 0.228 0.147 0.159 0.180 0.183 0.240 0.224 0.189 0.281 0.194 0.207 0.218 0.224 0.093 0.005 0.016

*Mean of three replications.

Table 5 Correlation between Cellulase, Polygalacturonase (PG) and Pectin methylesterase (PME) activity and root rot infection by Rhizoctonia bataticola isolates and their virulence pattern. Particular

Cellulase

PG

PME

RR infection

RR

0.934**

0.917**

0.633**

1.000

RR ¼ root rot infection %, ** ¼ highly significant, r ¼ @ 5% ¼ 0.433, 1% ¼ 0.549.

with the isolate Rb-32 (354.92 mg of D-glucose/ml extract), followed by Rb-33 (349.47 mg), Rb-40 (349.47 mg), Rb-3 (346.75 mg), Rb-10 (346.21 mg), Rb-13 (344.57 mg), Rb-23 (342.94 mg), Rb-6 and Rb38 (341.31 mg) and Rb-36 (340.22 mg) and uninoculated control recorded only 165.48 mg of D-glucose/ml of extract. There was less PG enzyme production even though some of the isolates were highly or moderately virulent and vice versa. There was a highly significant positive correlation (0.917**) between the PG enzyme activity and the virulence of the test isolates (Table 5). 3.3.3. PME activity PME activity of the root extracts of Soybean plants infected individually with twenty test isolates of R. bataticola was assayed by DNS method and the results (Table 4) indicated that the test isolates exhibited varied PME enzyme activity, which ranged from 0.147 to 0.281 OD/min. However, it was maximum (A620/min) with the isolate Rb-32 (0.281), followed by the isolates Rb-3 (0.272), Rb-24 (0.240), Rb-15 (0.228), Rb-27, Rb-40 (0.224), Rb-38, (0.218), Rb-36 (0.207) and Rb-1 (0.204) and it was lowest in untreated control (0.093 OD/min). There was less PME enzyme production even though some of the isolates were highly or moderately virulent and vice versa. There was a highly significant positive correlation (0.633**) between the PME enzyme activity and the virulence of the test isolates (Table 5).

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4. Discussion 4.1. Virulence of isolates Cell wall, composed of pectin, polysaccharides, cellulose, hemicellulose, lipids, and proteins, is degraded by the enzymes produced by a virulent pathogen [10]. There are reports that the more enzymes are produced by highly virulent isolates of corn than the weakly virulent ones [16]. In the present study, the results revealed that out of twenty R. bataticola isolates, seven isolates viz., Rb-10, Rb-13, Rb-15, Rb-17, Rb-21, Rb-23 and Rb-27 were moderately virulent and rest thirteen isolates viz., Rb-1, Rb-3, Rb-5, Rb-6, Rb-8, Rb-18, Rb-24, Rb-30, Rb-32, Rb-33, Rb-36, Rb-38 and Rb-40 were highly virulent. Maximum per cent root rot infection was recorded with the virulent isolates Rb-3, followed by Rb-33 and Rb32. Similar results were reported earlier by several workers [35,36]. Variations in the virulence of R. solani isolates were also reported. Pascual [37] reported that 52 isolates of R. solani belonged to anastomosis group AG1-1A which caused banded leaf and sheath blight in maize and showed considerable variations in their virulence. Mondal [36] reported 21 isolates of R. solani belonged to the relative lesion length and grouped into three categories; low, moderate, and highly virulent, which also exhibited variations in disease severity. 4.2. Production of extracellular enzymes by R. bataticola isolates (plate assay method) Cell-wall-degrading enzymes are thought to be important factors in determining the outcome of host-pathogen interactions [38]. However, little is known about the regulation of these enzymes in obligately biotrophic fungi. Pectinases are the first cell wall degrading enzymes secreted by pathogens and are also virulence determinant factors [20]. In the present study, 20 isolates of R. bataticola, inducing root rot in soybean were screened for their produce the enzymes viz., cellulase, pectinase, lipase, protease and amylase, by applying cupplate method. All fungal isolates were found to produce the enzymes cellulase, pectinase, lipase, protease and amylase, but to variable extent. R. bataticola isolates showed a wide variability in cellulase enzyme production, which evidenced by the formation of clear zone (mm) ranged from 19.00 to 71.00 mm. However, cellulase enzyme production was higher with the isolates Rb-33, Rb-36, Rb-38 and Rb-20, while it was lowest with the isolate Rb-5. The pectinase enzyme production by the test R. bataticola isolates ranged from 42.67 to 75.67 mm and it was higher with the isolates Rb-33, Rb-32, Rb-33, Rb-32 and Rb-13, while it was moderate with the isolates Rb-17 and Rb-27. Lipase enzyme production by R. bataticola isolates also varied from 40.33 to 82.33 mm and it was higher with the isolates Rb-10, Rb-13, Rb-6, Rb-38 and Rb-3, while it was moderate with the isolates Rb-17, Rb-27 and Rb-5. Protease enzyme production by R. bataticola isolates also varied from 47.67 to 85.33 mm and it was higher with the isolates Rb-32, Rb-38, Rb36, Rb-6, Rb-10, while it was moderate with the isolates Rb-8 and Rb-27. Amylase enzyme production by R. bataticola isolates ranged from 23.67 to 75.33 mm and it was higher with isolates Rb-36, Rb38, Rb-21, Rb-33 and Rb-32, while it was lowest with the isolates Rb-1, Rb-5, Rb-17 and Rb-27. In all of the uninoculated control plates, there was no any production of these extracellular enzymes. There was a highly significant positive correlation between the cellulose, pectinase, lipase, protease and amylase enzyme production and the virulence of the test isolates. Similar results on production of extracellular enzymes viz., amylase, lipase and protease production by haploids, diploids and heterocaryons of A. nidulans were reported [39]. For which, three

morphologically normal strains and 8 morphological mutants as well as various genetic combinations of the 11 strains were examined on solid culture medium containing specific substrate. For amylase and protease, the highest values were reached by some diploid and heterocaryons and for lipase by 1 morphological strain. The present results are also in agreement with Gopinath et al [40], who screened several fungal species for the production of extracellular enzymes such as amylase, protease, cellulase, and lipase. They correlated pathogenic ability of R. solani isolates with higher production of enzymes like pectic enzymes/PG and cellulose [41,42]. They also reported the association of cellulase and pectinase activities with virulence of indigenous Sclerotinia sclerotiorum isolates, in Jordan valley [43]. PG, polygalacturonase trans-eliminase and cellulase activities were reported directly correlated with the degree of virulence in Sclerotium bataticola inoculated sunflowers [44]. Boccas [45] found that, out of 248 fungal isolates recovered from coffee plants and soil samples and screened for their potential to produce pectinase, 119 isolates produced pectolytic enzyme, with 13 high producers, which were related to either Aspergillus or Penicillium. Dartora [46] found that out of 5 fungal strains tested for their capacity to produce endopolygalacturonase, strains of Aspergillus niger and A. oryzae were the best producers. Among 39 fungal isolates tested for their ability to produce pectinase enzyme on solid medium, only one isolate related to Emericella nidulans produced pectinase enzymes [47]. Lipases and proteases are very important enzymes in pathogenesis which attack the plasmalemma, after degradation of cell wall by protease along with pectolytic and cellulolytic enzymes. Like proteases, membrane degradation by lipases depends on the involvement of specific type of enzyme and membrane [48]. Stalk rot of corn was significantly correlated with cellulase production [49]. The role of enzymes produced by R. bataticola in the development of Arhar leaf spot/blight disease and penetration of the host tissue [50] and reported that the pathogen produced cellulolytic and pectolytic enzymes in vitro and in vivo. Eight fungal species representing 5 genera (positively infected broad bean plant) were assessed for their ability to produce pectinase enzyme using cup-plate method [51]. The enzyme activity of the fungal pathogens can be used as a predictive marker of pathogenicity and virulence of F. solani [52]. 4.3. Extracellular enzymes activity (plant sample analysis) Extracellular proteins secreted by fungus contains enzymes corresponding to the types of glycosidic linkages present in the host cell wall polysaccharides, which are implicated to macerate host tissues and degrade cell wall components [53]. The production and activity of pectinolytic and cellulolytic enzyme plays active role in disease development. The secretion of pectinase and cellulase enzymes by F. solani isolated from root rot infected C. forskohlii were noted [54]. Pectic enzymes are first polysaccharides to be induced by the fungi, when cultured on isolated plant cell wall or infected plant tissue [55]. In the present study, all twenty isolates of R. bataticola produced the enzyme viz., cellulase, PG and PME from the extract of Rhizoctonia-infected soybean root tissues, but with variable degrees. Result showed that the test isolates showed wide variability in cellulase enzyme activity in the range of 264.38e299.39 mg of Dglucose/hr/ml extract. However, cellulase activity was higher with the isolates Rb-1, followed by Rb-32 and it was lowest with the isolates Rb-5. PG enzyme activity ranged from 317.36 to 354.92 mg of D-glucose/ml extract. However, it was higher with the isolate Rb32, followed by Rb-33 and lowest with the isolate Rb-5. PME enzyme activity ranged from 0.147 to 0.281 OD/min. of root tissue. However, it was higher with the isolate Rb-32, followed by Rb-3 and least with the isolate Rb-17. There was a highly significant

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positive correlation between the cellulose, PG and PME enzyme production and the virulence of the test isolates of R. bataticola in Soybean. Similar results were reported indicating correlation between production of endo-PG and PME in vivo and virulence of isolates, which might also be playing an important role during pathogenesis [56]. PG production by R. bataticola was reported occur when both the culture and Rhizoctonia-infected tissues were 4e6 days old and the pH of the growth medium during this period ranged from 5.0 to 6.0 [57]. The growth, pH of the culture medium and the PG activity decreased after the 6th day's incubation. Dubey and Jain [58] evaluated extracellular enzymatic profiles of seven stone deteriorating fungi, among which A. niger, A. flavus, Eurotium amsteldomi and Alternaria sp. were reported to produce extracelluar enzyme activity. Acknowledgment The authors are thankful to the Dean, Post Graduate Institute and Head, Dept. of Plant Pathology and Agril. Microbiology, Mahatma Phule Krishi Vidyapeeth, Rahuri, Ahmednagar, Maharashtra, India, for the facilities provided to this works. References [1] S.T. Sonka, K.L. Bender, D.K. Fisher, Economics and marketing. p. 919e948, in: H.R. Boerma, J.E. Specht (Eds.), Soybeans: Improvement, Production and Uses, third ed., ASA, CSSA, and SSSA, Madison, WI, USA, 2004. [2] J.H. Hulse, Soybean: biodiversity and nutritional quality, in: Proceedings of the Second International Soybean Processing and Utilization Conference, 1996, pp. 377e381. Bangkok, Thailand. [3] J.R. Wilcox, World distribution and trade of soybean. p. 1e13, in: H.R. Boerma, J.E. Specht (Eds.), Soybeans: Improvement, Production, and Uses, third ed., ASA, CSSA, and SSSA, Madison, WI, USA, 2004. [4] Anonymous, All India Co-ordinated Research Project on Soybean, Directorate of Soybean Research Report and Summary Tables of Experiment, 2013-14, 2015. [5] S. Muthusamy, V. Mariappan, Disintegration of Sclerotia of Macrophomina phaseolina (Soybean isolate) by oil cake extracts, Indian Phytopath. 44 (1991) 271e273. [6] R.M. Cooper, The role of cell wall-degrading enzymes in infection and damage, in: R.K.S. Wood, G.J. Jellis (Eds.), Plant Diseases: Infection, Damage and Loss, Blackwell Scientific Publications, Oxford, 1984, pp. 13e27. [7] P.R. Keon, R.W. Byrde, R.M. Cooper, Some aspects of fungal enzymes that degrade plant cell walls, in: G.F. Pegg, P.G. Ayres (Eds.), Fungal Infection of Plants. Symposium of the British Mycological Society, Cambridge University Press, Cambridge, 1987, pp. 133e157. [8] S.O. Han, H. Yukawa, M. Inui, R.H. Doi, Regulation of expression of cellulosomal cellulase and hemicellulase genes in, Clostridium Cellulovorans. J. Bacteriol. 185 (2003) 6067e6075. [9] K. Keegstra, Plant cell walls, Plant Physiol. 154 (2010) 483e486. [10] H. Wheeler, Plant Pathogenesis, vols. 2e3, Acad. Press, New York & London, 1975. [11] C. Riou, G. Freyssinet, M. Fevre, Production of cell wall degrading enzymes by the Phytopathogenic fungus, Sclerotinia Sclerotiorum. Appl. Environ. Microbiol. (1991) 1478e1484. [12] W. Koller, C.R. Allan, P.E. Kolattukudy, Role of cutinase and cell wall degrading enzymes in infection of Pisilum sativum by Fusarium solani f. sp. pisi, Physiol. Pl. Pathol. 20 (1982) 47e60. [13] C.R. Howell, Use of enzyme deficient mutants of Verticillium dahliae to assess the importance of pectolytic enzymes in symptoms of Verticillium wilt of cotton, Phy. Pl. Path. 9 (1976) 279e283. [14] R.M. Cooper, R.K. Durrands, Selection, characterization, pathogenicity and virulence of pectinase-deficient mutants of Verticillium albo-atrum, in: E.C. Tjamos, C. Beckman (Eds.), Vascular Wilt Diseases of Plants, Springer Verlag, , Berlin, 1989, pp. 325e335. [15] M.J. Bailey, E. Pessa, Strain and process for production of polygalacturonase, Enzyme Microb. Technol. 12 (1990) 266e271. [16] E.A. Sadik, M.M. Payak, S.L. Mehta, Some biochemical aspects of hostpathogen interactions in Pythium stalk rot of maize. I. Role of toxin, pectolytic and cellulolytic enzymes in pathogenesis, Acta Phytopath. Acad. Sci. Hung. 18 (1983) 261e269. [17] A. Hameed, M.A. Natt, M.J. Iqbal, The role of protease and lipase in plant pathogenesis, Pak. J. Phytopathol. 6 (1994) 13e16. [18] W.M. Wanjiru, K. Zhensheng, H. Buchenauer, Importance of cell wall degrading enzymes produced by Fusarium graminearum during infection of wheat heads, Eur. J. Pl. Path. 108 (8) (2002) 803e810.

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