Biological Control of Leaf Blight of Wheat Caused by Bipolaris Sorokinian M.M

Bull. Inst. Trop. Agr., Kyushu Univ. 39: 43-51, 2016 43

Biological control of leaf blight of wheat caused by Bipolaris sorokinian M.M. Hossain1)*, I. Hossain2) and K. M. Khalequzzaman3)

Abstract Five different plant extracts including neem (Azadirachta indica), mehedi (Lawsonia alba), garlic clove (Allium sativum), rhizome of ginger (Zingiber officinales), seeds of black cumin (Nigella sativa), and BAU-Biofungicide (a Trichoderma based preparation) were used to evaluate the performance or effectiveness of those biological control agent on Bipolaris leaf blight of wheat and related pathogen (Bipolaris sorokiniana). Pathogenic reaction was observed in Bipolaris sorokiniana against different treatments by detached leaf method where leaf spot size was minimum (4.5mm) with BAU-Biofungicide and maximum leaf spot size (32.5mm) with control. Effect of seed treatment on wheat plant was evaluated by rolled paper towel method and BAU-Biofungicide, extracts of garlic clove and neem leaf at the value of 13%, 12% and 10%, respectively higher normal seedlings and BAU-Biofungicide also resulted 26.6% higher vigour index over control. Multiplication effect (seed treatment plus foliar spray of same treatment) of different treatments was examined to determine the efficacy for disease control. In pot and field experiments, though Bavistin and Tilt were most effective however, BAU-Biofungicide and extract of garlic clove were superior compared with the treatments used for controlling leaf blight of wheat. Multiplication effect of seed treatment plus foliar spray showed superior effect by BAU-Biofungicide including higher 1000-grain weight (43.92g) and grain yield (2.75 t/ha). Seed treatment with Bavistin and foliar spray with Tilt showed 1000-grain weight and grain yield by 47.12g and 3.0 t/ha, respectively. Our data also revealed that BAU-Biofungicide increased grain yield (29.87%) over control.

Keywords: Detached leaf, BAU-Biofungicide, AUDPC and extract

Introduction

Wheat (Triticum aestivum L.) is considered as one of the most important cereal crops in the world and it became to the second most important in Bangladesh. The average yield of this crop is only 2.60 ton/ha in Bangladesh (BBS, 2012) and damage caused by disease become to the main constraints of high yield. Leaf blight caused by Bipolaris sorokiniana of wheat is the most common and severe disease and the severity of the disease has been increased in an alarming proportion (Bazlur Rashid et al., 1987; Hossain and Azad, 1994; Alam et al., 1995). It causes foliar blight, seedling blight, head blight and common root rot of wheat (Hossain, 1991; Hossain and Khan, 1993; Hossain and Azad, 1994;

1) Senior Scientific Officer, Regional Agricultural Research Station, BARI, Jamalpur, Bangladesh 2) Professor, Department of Plant Pathology, BAU, Mymensingh, Bangladesh 3) Senior Scientific Officer, Spices Research Centre, BARI, Shibganj, Bogra, Bangladesh *Corresponding author: [email protected] 44 M.M. Hossain et al.

Shahzad et al., 2009). This disease is major constraint of wheat cultivation causing severe reduction of yield up to 40% and 88% over control, under natural field condition and artificial inoculation, respectively (Hossain et al., 1998). The high morphological, physiological and genetic variability makes the control of this fungus due to a difficult task (Poloni et al., 2009) and the yield loss of this disease ranged from 10-21% (Hossain and Azad, 1992; Malaker et al., 2004). The use of chemicals, has been found very effective in controlling fungal diseases of plant, but some major problems threaten to limit the continuous use of fungicides. Many research workers have tried to find out safe and economical control of plant diseases by using extracts of different plant parts (Akhter et al., 2006; Bdliya and Alkali, 2008). Plant extracts and biofungicide are also available to treat seed (Koike et al., 2011) In several studies’ it has been found that plant extract increased seed germination and vigour index in different crops after application of plant extracts (Hasan et al., 2004; Chowdhury et al., 2005a). The application of Trichoderma harzianum has been identified as potential biocontrol agents for the management of various crop diseases (Venn, 2011). This biocontrol agent has the potential to protect seedlings against several plant pathogenic fungi (Kucuk et al., 2007; Hasan and Alam, 2007). Trichoderma spp. is effective to reduce the foliar disease severity on wheat plants compared with untreated plants (Muthomi et al., 2007; Hasan and Alam, 2007; Sing et al., 2008). Hossain and Sultana (2011) stated that the BAU-Biofungicide effectively manage the seed and seedling diseases of maize. Biological control represents a natural and ecological approach for controlling diseases the reduce the usage of chemical inputs and their effects on environment. It is more stable and longer lasting than the controls and is comparative with the concepts and goals of integrated pest management and sustainable agriculture. Concerning to the health, safety and environmental effects of agricultural chemicals in our water, soil and food, the use of biological control need to be emphasized strongly. Biological control has now become one of the most exciting and rapidly developing areas in plant pathology, because it has great potential to solve many agricultural and environmental problems. Biological control has been proposed as a replacement for chemical control of plant diseases. However, the present study was under taken to develop a suitable biological control approach for the management of leaf blight of wheat with out use any chemicals.

Materials and Methods

Collection of seeds, plant materials and BAU-Biofungicide The seeds of wheat variety Kanchan (highly susceptible to leaf blight) were collected from Regional Agricultural Research Station (RARS), Jessore. Five plant materials including leaves of neem (Azadirachta indica), mehedi (Lawsonia alba), garlic clove (Allium sativum), rhizome of ginger (Zingiber officinales) and seeds of black cumin (Nigella sativa) were collected from Botanical garden of Bangladesh Agricultural University (BAU) and local market of Mymensingh. BAU-Biofungicide (a Trichoderma based preparation) was obtained from the Disease Resistance Laboratory of the Depart- ment of Plant Pathology, BAU, Mymensingh. Biological control of Bipolaris sorokiniana 45

Seed treatment Required amount of seeds presented each treatment with plant extracts (at 1% suspension), BAU- Biofungicide (at 2.5% seed weight) and Bavistin (at 0.1%) separately over night by dipping method and then separate the seeds by air dried. 1% suspension of plant extracts. Plant extracts and BAU-Biofungi- cide were prepared according to the method of Hossain and Azad (1994) and Hossain (2011). Excised leaf method and rolled paper towel method were performed the according to the method of Hossain and Schlosser (1993) and Singh and Rao (1997). The preparation of B. sorokiniana inoculum followed CIMMYT method (Gilchrist, 1985).

Sowing of seeds in pot Treated seeds were sown in pots (10 seeds /pot) at 2 cm depth in the soil with four replications. Irrigation and weeding were carried out and thinned to five seedlings. Preparation of pot soil was done following the method of Dasgupta, (1988). Experiments were conducted in the laboratory and experimental field of the Department of Plant Pathology, BAU, Mymensingh, Bangladesh. Completely randomized design with 4 replications in pot and randomized block design with three replications in field experiments were maintained. The plot size was 10 m2 and date of sowing was 6th December, 2011.

Application of spray materials Plant extracts (1%), BAU-Biofungicide (2%) and Tilt 250 EC (0.05%) were sprayed at 12 days inter- val commencing just after appearance of disease symptom.

Data collection and analysis Data on percent germination, percent abnormal seedlings, shoot and root length, yield and yield contributing characters were collected and analyzed. Vigour index (VI) was computed using the for- mula of Baki and Anderson (1973) as shown below: Vigor index= (Mean shoot length + Mean root length) x percent Germination Leaf blight severity was scored three times on double digit scale (00-99) reported by Saari and Prescott (1975) commencing from the water ripe to early dough stage (Zadoks et al. 1974). Disease data were converted to percent diseased leaf area (percent DLA) and then area under disease progress curve (AUDPC) was calculated according to the method of Sharma and Duveiller (2003).

% DLA = D1/9 X D2/9 X 100 where, D1 = First digit, representing relative disease height

D2 = Second digit, indicating disease severity on the foliage

AUDPCAUDPC == [(Y[(Yi+1i+ 1+ + Y Yi)i) xx 0.5]0.5] [[TTii+1+1 - Tii]] i=1 where, Yi = Disease severity at the ith observation,

Ti = Time (days) of the ith observation and n = Total number of observations (at least 3 observations). 46 M.M. Hossain et al.

Results and Discussion

Pot and field experiments were conducted to determine the combined effect of seed treatment plus flower spray with 5 plant extracts and BAU-Biofungicide in controlling leaf blight of wheat in vitro. Inhibition of pathogenic reaction caused by B. sorokiniana by using five plant extracts and BAU- Biofungicide were investigated following detached leaf method by artificial inoculation of wheat variety Kanchan with 6x104 conidia/ml in-vitro. No lesion was produced by the pathogen (B. sorokiniana) when Bavistin (0.1%) used on one and two month old seedlings. Only 4.5 mm lesion was produced in case of BAU-Biofungicide and 32.5mm in control (Table 1). The result of the experiment is in agree- ment with the findings of Galletti et al., (2008). Hossain and Ashrafuzzaman (1994) and Hossain et al (1997) reported that garlic clove and mehedi leaf completely inhibited the pathogenecity of B. sorokiniana on the excised wheat leaf. Effect of seed treatment with different plant extracts and BAU-Biofungi- cide on percent germination, percent normal seedlings and vigour index of wheat seeds were investi- gate. All treatments showed increased germination significantly over untreated control in pot and in- vitro conditions. Seed treatment with BAU-Biofungicide gave 95% germination in pot which showed at 9.0 % higher compared with the untreated control (Table 3). Biswas et al., (2008) reported that Tricho- derma treated rice seeds showed maximum germination (92%). Hossain et al. (2011) also stated that BAU-Biofungicide treated maize seeds increased the germination. Again, BAU-Biofungicide and botanicals showed significant effect on formation of normal seedlings which is due to antifungal effect of treatment. BAU-Biofungicide showed the best results producing the value at 15.83% higher normal seedlings compared with control, and higher percentage of abnormal seedling was also recorded in untreated control (Table 2). This result is in accordance with the findings of Hasan et al., (2005) and it is also supported by the reports of Chowdhury et al., (2005) and Raihan et al. (2003). Maximum (22.37) vigour index was also observed with BAU-Biofungicide and presented at 26.6 % higher than untreated control (Table 2). Similar results were observed by Shamsuzzaman et al., (2003),

Table 1. Effect of different plant extracts and BAU- Biofungicide on pathogenicity of Bipolaris sorokiniana causing leaf spot of wheat of one and two month old seedlings Treatment (dose) Lesion size (mm) after different period of time Leaf of one month old seedlings Leaf of two month old seedlings 1st count at 2nd count at 3rd count at 1st count at 2nd count at 3rd count at 48 HAI 72 HAI 96 HAI 48 HAI 72 HAI 96 HAI Garlic (1%) 7.25 (38.3) 15.50 (40.9) 22.50 (25.0) 10.50 (41.7) 17.50 (26.3) 27.50 (15.4) Ginger (1%) 9.25 (21.3) 18.75 (28.6) 23.25 (22.5) 15.50 (13.9) 21.25 (10.5) 30.00 (7.7) Kaligera (1%) 10.25 (12.8) 18.75 (28.6) 21.75 (27.5) 14.75 (18.1) 21.25 (10.5) 30.00 (7.7) Neem (1%) 11.00 (6.4) 25.75 (1.9) 27.50 (8.3) 15.75 (12.5) 19.50 (17.9) 28.75 (11.5) Mehedi (1%) 8.50 (27.7) 16.75 (36.2) 20.40 (32.0) 12.50 (30.6) 17.5 (26.3) 26.25 (19.2) BAU-Biofungicide (2.5%) 0.00 (100) 0.00 (100) 0.00 (100) 0.00 (100) 0.00 (100) 4.50 (86.2) Bavistin (0.1%) as check 0.00 (100) 0.00 (100) 0.00 (100) 0.00 (100) 0.00 (100) 0.00（100） Control (water) 11.75 26.25 30.00 18.00 23.75 32.50 LSD (0.05) * * * * * * CV (%) 5.29 9.72 8.34 7.81 10.57 6.19 HAI = Hours after inoculation Data represent the means of four replications Data in parentheses indicate percent decreased over control Biological control of Bipolaris sorokiniana 47

Hossain and Shamsuzzaman (2003) and Rao (2009). Several studies have demonstrated increasing seed germination and vigour index in different crops after application of plant extracts (Hasan et al., 2004; Chowdhury et al., 2005). Biswas et al. (2008) stated that T. harzianum treated rice seeds increased shoot and root length 21% and 25%, respectively.

Table 2. Effect of seed treatment with different plant extracts and BAU- Biofungicide on percent germination, vigour index, formation of normal and abnormal seedlings of wheat seedlings Treatment (dose) % Germination Vigour index % Normal % Abnormal (10 DAS) (10 DAS) seedlings seedlings Garlic (1%) 96.67 (+4.65) 20.93 (+18.45) 91.00 (+13.75) 9.00 (-55.00) Ginger (1%) 98.00 (+6.09) 21.03 (+19.01) 90.33 (+12.91) 9.67 (-51.65) Kalijera (1%) 94.33 (+2.12) 20.82 (+17.83) 89.33 (+11.66) 10.67 (-46.65) Neem (1%) 98.67 (+6.82) 19.47 (+10.19) 94.00 (+17.50) 6.00 (-70.00) Mehedi (1%) 96.00 (+3.92) 19.98 (+13.07) 90.00 (+12.50) 10.00 (-50.00) BAU-Biofungicide (2.5%) 97.33 (+5.36) 22.37 (+26.60) 92.67 (+15.83) 7.33 (-63.35) Bavistin (0.1%) as check 98.00 (+6.09) 19.01 (+7.58) 93.67 (+17.08) 6.33 (-68.35) Control (untreated) 92.37 17.67 80.00 20.00 LSD (0.05) * * * * CV (%) 8.90 20.15 6.81 5.13 DAS- Days after sowing Data represent the means of three replications Data in parentheses indicate percent increased (+) and % decreased (-) over control

By evaluating effects of seed treatment plus foliar spray of selected five plant extracts and BAU- Biofungicide on disease severity, grain yield and straw yield of wheat were varied in pot and field trials. The AUDPC value was minimum in case of Bavistin treated seeds plus Tilt-250EC sprayed pot (611.11 and 327.16) BAW-Biofungicide (827.16 and 388.88) and garlic clove (820.18 and 753.08) that also showed among the treatments. The treatment BAU-Biofungicide (seed treatment + spray) reduced a considerable disease severity as compared to untreated control in the present study. Similar result was observed by Biswas et al., (2008). Trichoderma spp. has been found to be effective in reducing the foliar disease severity on wheat plants compared with untreated plants (Sabatini et al., 2002; Perello et al., 2003; Muthimi et al., 2007; Hasan and Alam, 2007). Singh et al. (2008) conducted an experiment and proved that T. harzianum was effective in reducing the leaf blight incidence of wheat. Fattah et al. (2007) stated that spraying of spore suspension of T. harzianum at 108 spore/ ml significantly reduced the disease severity and disease incidence of rice brown spot caused by Bipolaris oryzae. Seed treatment plus foliar spray of BAU-Biofungicide, extract of garlic clove and mehedi leaf were increased grain yield by 29.87, 8.49 and 8.33%, respectively. The highest grain yield (3.0 t/ha) was recorded in case of Bavistin treated seeds plus Tilt spray followed by BAU-Biofungicide (2.8 t/ha) as seed treatment plus foliar spray. BAU-Biofungicide also increased the 1000-grain weitht of wheat at 43.92g whereas lowest (38.08g) was observed in untreated control (Table 4). Our data supported by Shultana et al. (2009). Hossain (2009) reported foliar spray of BAU-Biofungicide as most effective in reducing the leaf blight severity but significantly increased grain yield of wheat. Fattah et al. (2007) also stated that spraying of spore suspension of T.harzianum at 108 spore/ml significantly reduced the disease severity of rice brown spot caused by Bipolaris oryzae and also significantly increased the grain yield. 48 M.M. Hossain et al.

Table 3. Multiplication effect of seed treatment and foliar spray with plant extracts and BAU Biofungicide on yield and yield contributing characters of wheat Treatment (dose) % AUDPC Days to 1000-grain Grain yield Straw yield Seed Foliar spray germination maturity wt.(g) (g/pot) (g/pot) treatment Garlic (1%) Garlic (1%) 90.00 820.98 106 43.75 15.33 (15.87) 20.26 (5.79) Ginger (1%) Ginger (1%) 90.5 987.65 106 42.75 13.67 (3.32) 19.35 (5.87) Kaligera (1%) Kaligera (1%) 90.00 962.96 105 42.35 14.13 (6.80) 19.41 (5.95) Neem (1%) Neem (1%) 90.00 919.75 105 42.10 13.81 (4.38) 19.44 (5.97) Mehedi (1%) Mehedi (1%) 92.50 1043.2 105 43.50 14.33 (8.31) 20.33 (6.16) BAU- BAU- 95.00 827.16 108 46.50 17.05 (28.87) 23.79 (24.23) Biofungicide Biofungicide (2.5%) (2.0%) Bavistin Tilt (0.05%) 95.50 611.11 110 49.00 17.34 (31.06) 22.93 (19.74) (0.1%) as check Control 87.00 1209.87 105 42.00 13.23 19.15 LSD(0.05) * * * * * * CV (%) 7.66 18.67 7.76 11.27 15.37 8.11 Data represent the means of three replications Data in parentheses indicate the percent increased over control AUDPC = Area under disease progress curve

Table 4. Multiplication effect of seed treatment and foliar spray with plant extracts and BAU-Biofungicide on yield and yield contributing characters of wheat Treatment (dose) No. of AUDPC Days to 1000-grain Grain yield Straw yield effective maturity wt.(g) (kg/ha) (kg/ha) Seed Foliar spray 2 treatment tillers/m Garlic (1%) Garlic (1%) 252.0 753.08 108 39.76 2300.0 (8.49) 3133.3 (5.97) Ginger (1%) Ginger (1%) 253.0 1111.11 105 37.12 2151.2 (1.47) 3033.3 (2.59) Kaligera (1%) Kaligera (1%) 258.6 1018.51 107 38.80 2200.0 (3.77) 3300.0 (11.61) Neem (1%) Neem (1%) 250.6 993.82 105 40.74 2190.0 (3.30) 3300.0 (11.61) Mehedi (1%) Mehedi (1%) 254.3 987.65 105 39.96 2296.7 (8.33) 3100.0 (4.84) BAU- BAU- 266.0 388.88 110 43.92 2753.3 (29.87) 3950.0 (33.59) Biofungicide Biofungicide (2.0%) (2.0%) Bavistin Tilt (0.05%) 260.3 327.16 112 47.12 3000.0 (41.51) 3866.7 (30.78) (0.1%) as check Control 251.0 1043.2 104 38.08 2120 2956.7 LSD(0.05) * * * * * * CV (%) 12.31 20.86 8.32 8.31 14.27 13.09 Data represent the means of three replications Data in parentheses indicate the percent increased over control AUDPC = Area under disease progress curve Biological control of Bipolaris sorokiniana 49

Khan and Sinha (2005) stated that foliar spray of T. harzianum increased rice grain weight (20.25- 23.13%) and 1000-grain weight (6.36-7.35%). After using biocontrol agents’ the yield of winter wheat has increased by 160% and the 1000-grain weight by 4%. Tewari and Singh (2005) reported that foliar spray and soil treatment plus foliar spray of T. harzianum increased 1000-grain weight at 3.7% and 7.8%, respectively. BAU-Biofungicide showed maximum straw yield (3950kg/ha) followed by Tilt (3866.7kg/ha) i.e. 33.59% and 30.38%, respectively (Table 4). This finding is also supported by Shultana et al., (2009) and Hossain (2009). They recorded significant increase in straw yield using BAU-Biofungicide. From the results of Fig. 1 and 2, it is stated that the grain yield and straw yield of wheat were nega- tively correlated (r= 0.9106 *** and r= 0.8390 ***) with leaf blight severity (percent DLA). The result indicates that if disease score positively changed one unit, then grain yield and straw of wheat will be decreased 24.07 kg/ha and 27.43 kg/ha.

y = -24.077x + 3825.6 y = -24.077xR² = 0.8293 + 3825.6 3500 R² = 0.8293 3500 3000 3000 2500 2500 2000 2000

Yield (kg/ha) 1500

Yield (kg/ha) 1500 1000 1000 500 500 0 0 0 20 40 60 80 100 0 20 40 60 80 100 Disease severity (% DLA) Disease severity (% DLA) Fig. 1. Relationship between disease severity and yield of wheat cv. Kanchan Fig. 1. Relationship between disease severity and yield of wheat cv. Kanchan Fig. 1. Relationship between disease severity and yield of wheat cv. Kanchan

y = -27.432x + 4993.7 y = -27.432xR² = 0.704 + 4993.7 R² = 0.704 4500 40004500 35004000 30003500 25003000 20002500 15002000 10001500 Straw yield (kg/ha) yield Straw 1000 Straw yield (kg/ha) yield Straw 500 5000 0 0 20 40 60 80 100 0 20 40 60 80 100 Disease severity (% DLA) Disease severity (% DLA) Fig. 2. Relationship between disease severity and straw yield of wheat cv. Kanchan Fig. 2.Fig. Relationship 2. Relationship between diseasebetween severity disease and severity straw yield and of straw wheat yield cv. Kanchan of wheat cv. Kanchan 50 M.M. Hossain et al.

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