Prevalence of Cucumber Mosaic Virus and Its Impact on Growth and Yield of Different Chili Cultivar M

Bull. Inst. Trop. Agr., Kyushu Univ. 39: 65-74, 2016 65

Prevalence of Cucumber Mosaic Virus and its impact on growth and yield of different chili cultivar M. S. Rahman1)*, M.S. Akhter1), M.M. Alam1), N. Pervin1) and A. M. Akanda2)

Abstract With a view to determine prevalence and impact of Cucumber mosaic virus (CMV) on chilli (Capsicum annum L.) eleven varieties consisting one Bangladesh Agricultural Research Institute (BARI) released variety BARI Mar- ich-1, and ten local varieties like Chittagong, Comilla-1, Jamalpur, Gazipur, Chandpur, Pusa jawla, Comilla-2, Kus- tia, Bogra and Balujhuri were tested. The results of the symptomatological study, host range test and Double Anti- body Sandwich Enzyme-Linked Immune - Sorbent Assay (DAS-ELISA) revealed that the identified virus was Cucumber mosaic virus (CMV). The incidence of CMV was recorded as 3.00 to 21.21% depending on the varieties. The highest prevalence (21.21%) of CMV was recorded in Kustia and lowest (3.00%) was in variety Comilla-2. Sig- nificant (P=0.05) reduction of growth and yield contributing characters of different chilli varieties were observed due to CMV infection. The highest plant height (39.21%) and canopy diameter/spreading (36.84%) reduction were recorded in variety Chandpur and Balujhuri respectively and lowest plant height (15.67%) and canopy diameter (8.88%) reduction were recorded in variety Comilla-2. The number of fruit/plant reduction was varies from 49.63 to 89.29% where highest in variety Balujhuri and lowest in Comilla. Fruit length and fruit weight reduction were found from 14.03 to 44.14% and 10.56 to 36.96%, respectively.

Keywords: Chilli, CMV, Prevalence, Serology, DAS-ELISA

Introduction

Chilli (Capsicum annum L. Family: Solanaceae), is an important commercial spice crop widely cultivated all over the world. It is one of the richest sources of vitamin C and A (Howard, 2000). Chilli fruits are consumed as fresh, dried or processed products, table purposes as well as spice and condi- ments. In Bangladesh, 2009 cropping season, the crop is cultivated in an area about 89268 ha with a total production of 109337 tons (FAOSAT, 2011). Chilli is a common spice crop growing in all seasons and areas. This crop is grown in kitchen garden as well as commercially in the field and it occupies the second position next to onion. Chilli plants are easily infected by fungi, bacteria and virus which cause severe damage. Among these pathogens, plant viruses are particularly difficult to control and are estimated to cause economic losses of about 15 billion US dollars per annum worldwide (van Fanbing, 1999). Approximately 50 spe-

1) Scientific officer, Bangladesh Agricultural Research Institute, Bangladesh 2) Professor, Department of Plant Pathology, BSMRAU, Gazipur-1706 *Corresponding author: [email protected] 66 M. S. Rahman et al. cies of pepper viral diseases have been reported in the world (Green and Kim, 1991). However, Brunt et al. (1990) reported that most commonly recorded viruses of chilli are Cucumber mosaic virus (CMV), Potato virus Y (PVY), Pepper mottle virus (PMV), Tomato spotted wilt virus (TSWV) and Tobacco mosaic virus (TMV). Moreover, among the virus diseases of chilli, CMV has been reported the most economically important aphid-borne virus having wide host rage including herbaceous and woody plants all over the world (Francki et al. 1979). In Bangladesh, Akanda et al. (1991) reported the occurrence of CMV in Solanaceous and Cucurbitaceous plants. As the viral diseases are difficult to control, it is neces- sary to develop field tolerant variety having multiple- disease resistance gene (Grube et al. 2000; Monma and Sakata 1997). In spite of the commercial importance of chilli in Bangladesh, scanty works have been done to find out CMV resistance source. Therefore, the present investigation was undertaken to test the available cultivars of chilli against CMV to identify the source of resistance.

Materials and Methods

The field experiment was conducted at the research farm of Bangabandhu Sheikh Mujibur Rahman Agricultural University (BSMRAU), Gazipur. The soil of the experimental field belongs to Salna series under the Agroecological Zone (AEZ)-28: Madhupur Tract. The pH of the soil ranges from 6 to 6.5. Ten local cultivars namely Chittagong (V2), Comilla-1 (V3), Jamalpur (V4), Gazipur (V5), Chandpur (V6), Pusa jawla (V7), Comilla-2 (V8), Kustia (V9), Bogra (V10) and Balujhuri (V11) and one Bangladesh Agricultural

Research Institute (BARI) released variety BARI Marich-1 (V1) were tested in this experiment. The experiment was conducted using Randomized Complete Block Design (RCBD) with four replications.

Symptomatological study: Initially the virus was identified by Symptomatological study through visual observation of typical symptoms of CMV developed on naturally infected chilli plants (Green and Kalloo 1994). The symptoms appeared in the naturally infected chilli plants were closely and carefully observed. The symptoms developed in the foliar parts of the plants including leaves, flowers and fruits were noted.

Double Antibody Sandwich Enzyme-Linked Immune–Sorbent Assay (DAS-ELISA): DAS-ELISA was employed for detecting the CMV from naturally infected chilli plants collected from field as well as inoculated plants following outline of Clark and Adams (1977) with slight modification as suggested by Akanda et.al (1991). The sap was extracted from the leaf samples in extraction buffer at l: 10 ratios (tissue weight: extraction buffer volume). The extracted sap was poured at 100μl per well of the ELISA plate which was pre coated with virus specific IgG. The plate was then incubated in a humid box at room temperature for 2 hr. After incubation, the plate was washed with washing buffer and then enzyme conjugate was dispensed at 100μl per well. The plate was incubated and washed following the same procedure as mentioned before. The substrate solution was prepared following the instructions in the manual and used at 100μl per well. The plate was incubated in a humid box at room temperature for 60 min. just after 60 min of incubation, 50μl of 3M sodium hydroxide was added to each well to stop the reaction. The plate was then observed visually to detect the development of yellow color in case of Prevalence of CMV & its impact on chilli 67 positive reaction. The optical density (OD) values were measured with ELISA reader EAR00 FW (SLT- LAVINSTRUMENTS) at 405 nm wavelength.

Mechanical inoculation test: The mechanical inoculation test was carried out following the method described by (Hill 1984). Three different plant species were used in the test. The plant species were Capsicum annum, Chenopodium amarenticolor and Datura stramonium. The plants were grown in an insect proof net house using five inches earthen pot. The soils used in the pot were amended with manure and fertilizer as per recommendation (Akanda, 1991). Three different buffers namely 0.01M potassium phosphate buffer, pH 7.1, sodium phosphate buffer, pH 7.1, and sodium potassium phosphate buffer, pH7.1, were used to extract sap of infected chilli leaves in the inoculation test. In each case, 5g fresh leaves collected from infected chilli plants were macerated using 50ml aforesaid buffers for sap extraction. The leaves of the test plants dusted with sterilized 600 mesh carborumdum powder. The sap was then rubbed with finger onto the carborundum powder dusted leaves of the test plants. After rubbing with sap, the inoculated leaves were carefully washed with sterilized water using wash bottle. The inoculated plants were placed in insect proof net house and were checked everyday to detect the appearance of symptoms.

Disease incidences Disease incidences were calculated by using the following formula:

X Diseases incidence (%) = 1 100 X2

Where, X1= No. of infected plants, X2= Total no. of plants

Growth and yield attributes: Five healthy plants of each plot were randomly selected and tagged to collect data for growth and yield attributes of plant height, spreading, fruits/plant, fruit weight and fruit length. Similar data were recorded separately from CMV infected plants. Spreading (cm) of plants was measured by average length of North-South and East-West coverage of fully matured chilli plants in both cases (healthy and infected).

Percent reduction Percent reduction of the growth and yield contributing characters were calculated by using the following formula:

Y Y R = 1 100 Y

Where, R = Percent reduction of growth/ yield contributing character, Y = Growth/ yield contributing characters of healthy plants, Y1 = Growth/ yield contributing characters of infected plants. Data were analyzed by analysis of variance (ANOVA) using MSTAT-C program and means were compared according to Duncan’s Multiple Range Test (Gomez and Gomez 1984). 68 M. S. Rahman et al.

Results and Discussions

Symptomatological study of CMV: Mild mosaic and mottling, shoe string, fern leaf, vein bending, vein clearing and leaf deformation was characteristically observed as prominent symptoms in the naturally infected chilli plants (Fig. 1). The infected leaves become deformed in size and the mosaic symptoms turn into yellow at the later stage. The branches of infected plants became thinner as compared to healthy one. Growth retardation resulting stunting of plants was observed. The infected fruits become malformed and reduced in size. Some fruits become smaller and thinner as compared to healthy ones. Some fruits were observed curved shape and shriveled. All these symptoms observed in the naturally infected plants were typical viral disease-like symptoms. Moreover, the symptoms in infected chili plants seemed to be identical to CMV as described by Suzuki et al. (2003)

A B

C D

Fig.1. Variable symptoms induced by CMV in chilli. (A) Mosaic and vein clearing, (B) Shoe string and vein banding, (C) showing shoe string, vein banding and vein clearing, (D) Fern leaf . Fig.1. Variable symptoms induced by CMV in chilli. (A) Mosaic and vein clearing, (B)

Shoe string and vein banding, (C) showing shoe string, vein banding and vein clearing,

(D) Fern leaf . Prevalence of CMV & its impact on chilli 69

Serological identification of CMV by DAS-ELISA: Fourteen samples from experimental field were tested, 5 samples (about 35%) were reacted positively against the antiserum of CMV. The rest of the samples did not show any reaction with the antiserum. The positively reacted samples in DAS-ELISA produced bright yellow color (Bos 1969, Noordam 1973). The result indicated that the collected 14 samples which showed viral disease-like symptoms in the field, showed negative in DAS-ELISA may have been infected with other viruses of chilli. Plants appearing symptoms but negative in DAS-ELISA might be due to infection of virus for which antiserum was not used, abiotic agents or plant were suf- fering from nutritional or physiological disorders (Bos 1983).

Mechanical inoculation test: The mechanical transmissible capacity of the CMV positive samples were tested to the leaves of chilli plants. The CMV positive samples produced shoe string, mosaic with vein bending, leaf deformation and stunting symptoms. Chlorotic local lesions were developed in inoculated leaves of Chenopodium amaranticolor plants when inoculated with the naturally infected sample of chili. Chlorosis and systemic mosaic occurred in the leaves of Datura stramonium after inoculation with the naturally infected sample of chilli. Detail studies of CMV indicate that the virus generally produced shoe string, mosaic with vein banding, leaf deformation, fern leaf, vein clearing and stunting symptoms in any stage of growth. However, shoe string, vein banding, mottling, stunting, leaf deformation were the most common symptoms of CMV isolates in artificially inoculated plants.

Prevalence of CMV on different chilli varieties: Among the eleven chilli varieties, CMV incidences under field condition were recorded ranged between 3.00% to 21.21% (Table 1). The highest disease incidence was recorded in Kustia (21.21%) followed by Gazipur (17.53 %). Lowest disease incidence was observed in Comilla -2 (3.00%) followed by Bogra (7.00%). Choi et al., (2005) conducted a survey on pepper virus diseases in 31 regions in Korea and concluded that CMV was the most predominant virus in all inspected fields.

Table 1. Incidence, OD value and symptoms of Cucumber mosaic virus (CMV) on naturally infected different chilli cultivars. Variety Disease incidence a (%) OD value (A405nm)b Symptoms c

BARI Marich-1 (V1) 15.00(22.78) abc 0.96 St,Fl

Chittagong (V2) 14.54(22.41) abc 0.76 Vb,St,Fl

Comilla-1 (V3) 11.00(19.36) abc 0.80 Fl,V b,Vc

Jamalpur (V4) 12.12(20.37) abc 0.68 Mo,Vc

Gazipur (V5) 17.27(24.55) ab 0.97 Ld,St,Fl

Chandpur (V6) 9.08(17.53) bc 0.19 Mo,V b

Pusa jawla (V7) 7.04(15.38)c 0.39 Gm,Sm,Ld,St

Comilla-2 (V8) 3.00(9.97)d 0.27 Less St,Mo

Kustia (V9) 21.12(27.35)a 1.80 Gm,Sm,Ld,Vb

Bogra (V10) 7.00(15.34) c 0.45 Fl,Vc

Balujhuri (V11) 13.12(21.23) abc 0.64 Vb,Gm,St a Average of four replications in field condition in two consecutives years, b Means of three wells, OD value of buffer and healthy control are .03 and .07 respectively c Symptoms of CMV on chilli cultivars, St: Shoes string, Fl: Fern leaf, Vb: Vein banding, Vc: Vein clearing, Ld: Leaf distortion, Gm: Green motling, Mo: Mosaic, Sm: Severe mosaic 70 M. S. Rahman et al.

Effect of Cucumber mosaic virus (CMV) on growth attributes of different chilli varieties Significant reduction of plant height and spreading (canopy diameter) were observed in different chilli varieties due to CMV infection (Table 2, Fig 2). The highest plant height reduction was found in

Chandpur (39.21%) which was statistically identical with Balujhuri (38.82%), Kustia (36.41%), Gazi- pur (34.34%) and Pusa jawla (32.78%). The lowest plant height reduction was recorded in Comilla-2 (15.67%) followed by BARI Chilli-1 (18.40%) and Comilla-1 (19.47%). Plant height reduction due to CMV infection in Bogra (23.73%) was statistically identical with BARI Chilli-1 (18.40%) and Comilla-1 (19.47%). Fig. 2 represented that the trend of plant height reduction in comparison to healthy plant. The highest plant height reduction was in Chandpur and lowest in Comilla-2. The highest canopy diameter reduction was recorded in Balujhuri (36.84%), followed by Pusa jawla (32.05%), BARI-chilli1 (31.58%) and Gazipur (30.14%). The lowest canopy diameter reduction was observed in Comilla-2 (8.88%) followed by comilla-1 (12.61%) in Table 2. All the eleven chilli varieties infected by CMV were showed significant reduction of growth as compared to healthy plants.

Table 2. Effect of Cucumber mosaic virus (CMV) on growth and yield of different chilli cultivars percentage reduction Variety Plant height Spreading Fruit number Fruit length Fruit weight

BARI Marich-1 (V1) 18.40 ef 31.58 ab 83.11 ab 33.85 b 32.75 ab (25.14) (31.94) (66.33) (35.53) (34.80)

Chittagong (V2) 30.48 bcd 19.08 cd 79.13 ab 33.88 b 36.96 a (33.15) (25.41) (62.83) (35.50) (37.21)

Comilla-1 (V3) 19.47 ef 12.61 e 61.27 cd 40.19 ab 24.19 cd (25.83) (19.50) (51.54) (39.34) (29.30)

Jamalpur (V4) 27.99 cd 18.29 cd 75.02 abc 33.59 b 25.27 bcd (31.90) (24.76) (60.06) (35.42) (30.11)

Gazipur (V5) 34.34abc 30.14 ab 82.63 ab 26.15 c 22.16 d (35.29) (32.99) (65.79) (30.53) (27.90)

Chandpur (V6) 39.21 a 27.47b 76.71 abc 40.16 ab 29.95 abcd (38.75) (31.51) (61.27) (39.32) (33.13)

Pusa jawla (V7) 32.78 abc 32.05 ab 71.76bc 44.14 a 27.70 bcd (34.89) (33.45) (57.95) (41.64) (31.74)

Comilla-2 (V8) 15.67 f 8.88 e 49.63 d 14.03 d 10.56 e (23.15) (16.50) (44.94) (21.98) (18.49)

Kustia (V9) 36.41 ab 24.53 bc 76.55 abc 23.30 c 23.29 d (37.07) (29.41) (61.27) (28.62) (28.42)

Bogra (V10) 23.73 de 13.18 de 69.06 bc 21.98 c 24.68 bcd (29.08) (20.88) (56.91) (27.64) (29.71)

Balujhuri (V11) 38.82 a 36.84 a 89.29a 40.29 ab 32.07 abc (38.21) (37.21) (71.00) (39.39) (34.26) CV% 9.57 12.00 11.08 8.60 10.81 # Average of ten plants of two consecutives cropping season, *Means followed by same letters in row and column are not significantly different at 5% level by DMRT. Data given in the parenthesis are transformed value. Prevalence of CMV & its impact on chilli 71

Healthy Infected %Reduction 90 45 80 40

70 35 )

m n c o ( 60 30 i

t t c h u g 50 25 i d e e h

t 40 20

) n a l %

30 15 ( P 20 10 10 5 0 0 V1 V2 V3 V4 V5 V6 V7 V8 V9 V10 V11 Variety

FFig.ig .2. 2. P lPlantant h eheightight of ofhe ahealthylthy and and CM CMVV inf einfectedcted chil lchillii plan plantt and panderc epercentagentage of re dofuc reduction - tion of plant height by CMV. Vertical bar indicate the LSD value at 5% level of of plant hsignificance.eight by CMV. Vertical bar indicate the LSD value at 5% level of significance.

Effect of Cucumber mosaic virus (CMV) on yield attributes of different chilli varieties Due to CMV infection, all varieties under field condition showed significant reduction of fruits as well as fruit length and weight which was shown in Table 2 and Fig. 3, 4 and 5. The highest fruit number reduction was recorded in (89.29%) which was statistically similar with BARI Marich-1 (83.11%), Gazipur (82.62%), Chittagong (79.13%), Chandpur (76.71%), Kustia (76.55%) and Jamalpur (75.02%). The lowest fruit number reduction was recorded in Comilla-2 (49.63%) followed by Comilla-1(61.27%). The results rom the Fig. 3 it was revealed that highest number of fruits/plant was yielded by Comilla-2 whereas lowest number of fruits/plant was observed in Kustia. The highest fruit length reduction was

Healthy Infected %reduction 350 100

t 90 n

a 300 l p /

t 80 i u

r f

n f 250 o o

70 i t s c r e

b 60 ed u

m 200 R

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50 % ( 150 40

100 30

20 50 10

0 0 V1 V2 V3 V4 V5 V6 V7 V8 V9 V10 V11

Variety

Fig. 3. Numbers of fruit of healthy and CMV infected chilli plant and percentage Fig.3. Nureductionmbers of fofru ifruitt of h numbersealthy an dby C MCMV.V in fVerticalected ch ibarlli p indicatelant and ptheerce LSDntag valuee at 5% level of significance. reduction of fruit numbers by CMV. Vertical bar indicate the LSD value at 5% level of

significance. 72 M. S. Rahman et al.

Healthy Infected %reduction 9 50

8 45

40 7

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c 6

( n

o h i t 30 t g c n 5 u e d l

e t i

25 R u

r ) F

4 % ( 20 3 15

2 10

1 5

0 0 V1 V2 V3 V4 V5 V6 V7 V8 V9 V10 V11

Variety

Fig. 4. Fruit length of healthy and CMV infected chilli plant and percentage reduction of fruit length by CMV. Vertical bar indicate the LSD value at 5% level of significance. Fig.4. Fruit length of healthy and CMV infected chilli plant and percentage reduction Healthy Infected %reduction 2.5of fruit length by CMV. Vertical bar indicate the LSD value at 5% level o4f0

significance. 35 2

)

t 30 i u r

f / n g o i ( t

25 c 1.5 gh t i ed u e R w

i 20 u % ) r ( F 1 15

10 0.5 5

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Variety

Fig. 5. Fruit weight of healthy and CMV infected chilli plant and percentage reduction of fruit weight by CMV. Vertical bar indicate the LSD value at 5% level of significance. Fig. 5. Fruit weight of healthy and CMV infected chilli plant and percentage reduction of

fruit weight by CMV. Vertical bar indicate the LSD value at 5% level of found in Pusajawla (44.14%) which was statistically similar with Balujhuri (40.29%), Comilla-1 (40.19%) and Chandpursigni f(40.16%)icance. Table 2. The highest fruit length and fruit weight was recoded in Pua- jawla and Balujhuri respectively (Fig. 4 and 5), while due to CMV infection, lowest fruit length and fruit weight reduction were recorded 14.03% and 10.56% in Comilla-2 respectively (Table 2. and Fig. 4 and 5). The highest fruit weight reduction was recorded in Chittagong V2 (36.96%) which was statistically identical with BARI chilli-1 (32.75%), Balujhuri/ V11 (32.07%) and Chandpur (29.95%). The results Prevalence of CMV & its impact on chilli 73 proved that CMV caused remarkable yield loss in different chilli varieties except Comilla-2 which is seemed to tolerant against CMV infection. Therefore, Comilla-2 can be used as a resistance source for chilli breeding in the future.

Acknowledgement

The author humbly expresses his gratitude to the Director General & Director Training and Commu- nication, Bangladesh Agricultural Research Institute (BARI), Joydebpur, Gazipur for their time to time administrative support for his study. The author also expresses their gratitude to the Project Director (Spices Research Centre) for financial support.

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