Authors: Fatema Begum1, M. A. T. Masud2, A. M. Akanda3, I. H. Mian3


Fatema Begum,   M. A. T. Masud, A. M. Akanda, and I. H. Mian. 2014. Response of pumpkin breeding lines to Papaya Ringspot Virus-W. Bangladesh J. Plant Pathol. 30(1&2):39-44.

A field experiment was conducted under field condition to screen 29 breeding lines of pumpkin against Papaya ring spot virus-W (PRSV-W) under artificially inoculated condition. Five lines (Pk67-1-9-10, Pk13-1-1-9, Pk01-10-9-4-7, BARI mistikumra 1 and Pk102-5) showed characteristic mosaic symptoms of PRSV-W infection. Seventeen lines (Pk05-4-1-1, Pk67-1-9-10, Pk67-1-9-6, Pk37-1-4-6,  Pk13-1-1-9, Pk20-2-1-9, Pk02-2-1-6, Pk55-2-2-10, Pk61-1-1-5, Pk54-4-12-9, Pk54-4-12-1, Pk05-1-2-4, Pk05-1-2-10, Pk01-10-9-4-7, BARI mistikumra 1, Pk102-5 and Pk105-2) manifested positive reaction in ELISA to PRSV-W infection. Of these, 7 lines showed resistant reaction having disease incidence of 13.3 to 24.4% and 12 lines exhibited negative reaction to PRSV-W which was graded as highly resistant. Maximum disease incidence (58.3%) was recorded on Pk67-1-9-10 while minimum on Pk13-1-1-9 (9.0%). Significantly the highest disease severity and AUDPC were found on line BARI mistikumra 1. The disease severity ranged 13.3-15.0% and AUDPC ranged 46.7-52.5 in three lines (Pk13-1-1-9, Pk20-2-1-9 and Pk02-2-1-6). Pumpkin lines which showing highly resistance and resistance reactions to PRSV-W may be used in breeding program for pumpkin improvement program.


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At least 35 viruses infect cucurbits including pumpkin (Lovisolo 1980). Among the viruses Papaya Ringspot Virus – Water melon strain (PRSV-W) is the most destructive one causing significant reduction in plant growth and yield (Rezende and Pacheco 1998). The virus was first described in papaya (PRSV-P) by Jensen (1949) and in cucurbits (PRSV-W) by Webb (1965) and Webb and Scott (1965). Akanda (1991) reported the virus from Bangladesh and he found that it may cause 70-100% yield reduction of cucurbits depending upon the stage of infection. PRSV-W belongs to the family Potyviridae and is transmitted by aphids in a non-persistent manner (Purcifull et al. 1984).

Major virus control strategies include the use of insecticides to eliminate virus vectors, herbicides to remove alternative hosts for the virus and genetic resistance (Provvidenti 1993), which is often pathogen-specific (Grumet 1989). Of those control strategies, the most economical method is genetic resistance. Virus resistance may also be accomplished through virus coat proteins transferred into existing cultivars (Namba et al. 1992, Quemada et al. 1990), or by screening of germplasm.

The present investigation was undertaken to determine the response of pumpkin lines to PRSV-W and to determine the effect of PRSV-W on plant growth and yield.


A field experiment was conducted to screen 29 pumpkin lines against PRSV-W under artificially inoculated condition. The experiment was conducted in the experimental field of Bangabandhu Sheikh Mujibur Rahman Agricultural University (BSMRAU), Gazipur during October 2011 to April 2012 to screen. PRSV-W infected pumpkin plants of previous year experiment were selected on the basis of visible symptoms and Enzyme-Linked Immuno Sorbent-Assay (ELIZA). Leaf samples were collected from the selected PRSV-W infected plants. Inoculum of PRSV-W was prepared by grinding the infected leaves using mortar and pestle in 0.02 M phosphate buffer, pH 7.0. Leaf sample to buffer ratio was 1:10 (1 g infected leaf and 10 ml buffer). The sap of infected pumpkin leaves obtained after passing through double-ply cheese cloth was used as inoculum. Mechanical inoculation method using carborundum powder (800 meshes, Fisher Scientific, Fair Lawn, NJ) was followed (Daryono 2006).

Seedlings of the selected lines were raised in polyethylene bags. Before development of the true leaf, both cotyledons of seedlings were rubbed with carborundum powder to make minor injuries.

The inoculum sap was soaked with cotton and gently rubbed on the injured areas of leaf. After inoculation, carborundum powder was washed off with sterilized distilled water. Inoculated pumpkin seedlings were kept in aphid-proof cages for 10 days and transplanted in the main field. Standard procedures were followed for cultivation of land, preparation of bed and pits, application of manures and fertilizers, transplanting of seedlings, intercultural operations (Razzaque et al. 2000).

Pumpkin plants grown in the experimental field were checked at 55 days after transplanting to record the incidence of PRSV-W.  Disease incidence was identified based on visible symptoms followed by serological test using PRSV-W antiserum (Wei et al. 2001). The lines which showed positive reaction to PRSV-W antiserum were graded as susceptible and those showed negative reaction to the antiserum were graded as resistant according to Daryono (2006).

Data on disease incidence, disease severity and area under disease progress curve (AUDPC) of virus disease in experimental field were recorded through frequent visit after appearance of symptoms. Disease incidence was estimated using a standard formula (Agrios 2005):response-of-pumpkin-breeding-lines-to-papaya-ringspot-virus-w

According to Begum and Khan (1996), the lines were graded based on degree of disease incidence as highly resistant (HR=0.0% disease incidence), resistant (R=0-25% disease incidence), moderately resistant (MR= 26-50% disease incidence), moderately susceptible (MS= 51-75% disease incidence) and susceptible (S= 76-100% disease incidence). Disease severity was expressed in percent disease index (PDI). The PDI was computed using a standard formula (Piper et al. 1996) as shown below:


The severity of virus disease of pumpkin was indexed on a 0-5 indexing scale, where   0 = no visible symptoms, 1 = slightly mosaic on leaves, 2 = mosaic patches and/or necrotic spots on leaves, 3= leaves near apical meristem deformed slightly, yellow, and reduced in size; 4= apical meristem with mosaic and deformation, and 5= extensive mosaic and serious deformation of leaves (Xu et al. 2004).

Area under the disease progression curve (AUDPC) wad calculated according to Tooley and Grau (1984) using the formula shown below:

AUDPC=∑ ([xi+xi+1)/2] (ti+1-ti), where, Xi= cumulative disease incidence, expressed as a proportion of observation, ti= time (days after planting) the ith observation and n= total no. of observation.

The experiment was laid out following randomized complete block design with three replications. Each replication was consisted of four plants. Data con growth parameters (branches/plant and vine length), yield (relative to fruits/plant, yield/plant) and total soluble solids (%TSS) were recorded. Correlation of plant growth parameters and yield with PRSV-W incidence determined using MSTAT-C software and mean separation was done by DMRT.


Development of symptoms of PRSV-W in inocu-lated plants

Upon inoculation, pumpkin lines tested in the present experiment exhibited various degrees of symptoms of PRSV-W infection such as mosaic, vein clearing, leaf curling, blistering, deformation of the leaf leading to the formation of fern leaf or shoe string. Mild mosaic with vein clearing and vein banding were also observed on infected leaves as associated symptoms. At later stage of disease development, entire leaves became deformed and small. The infected plants produced deformed, smaller fruits having green raised spots scattered on the surface. The older leaves were small and deformed fern leaf like appearance (Plate 1).

Reaction of pumpkin lines to PRSV-W

On inoculation of pumpkin leaves with PRSV-W, lines Pk67-1-9-10, Pk13-1-1-9, Pk01-10-9-4-7, BARI mistikumra 1 and Pk102-5 showed mosaic symptoms at cotyledon stage. Symptoms developed after cotyledon stage, were severe mosaic on the young leaves, crinkling and blisters on the older leaves (Plate I). ELISA test was performed using one antiserum (PRSV-W) to detect presence of PRSV-W in inoculated leaves showing no visible symptoms of virus infection.

Out of 29 pumpkin lines tested, 17  (Pk05-4-1-1, Pk67-1-9-10, Pk67-1-9-6, Pk37-1-4-6, Pk13-1-1-9, Pk20-2-1-9, Pk02-2-1-6, Pk55-2-2-10, Pk61-1-1-5, Pk54-4-12-9, Pk54-4-12-1, Pk05-1-2-4, Pk05-1-2-10, Pk01-10-9-4-7, BARI mistikumra 1, Pk102-5 and Pk105-2) showed positive reaction against PRSV-W antiserum. These were graded as susceptible. Remaining 12 lines did not show any visible symptoms of PRSV-W infection or positive reaction against PRSV-W antiserum and graded as resistant to the virus.

Disease incidence, severity and AUDPC

Significantly the highest incidence PRSV-W (54.3-58.3%) was recorded from pumpkin lines Pk67-1-9-10, BARI mistikumra 1, Pk67-1-9-6, which were graded as moderately susceptible (MS). In lines Pk105-2, Pk37-1-4-6, Pk05-1-2-10, Pk102-5, Pk05-4-1-1 and Pk05-1-2-4, the disease incidence was 26.0-46.3% and these were graded s moderately resistant (MR). The incidence of PRSV-W was 9.0-24.3% on lines Pk01-10-9-4-7, Pk02-2-1-6, Pk55-2-2-10, Pk61-1-1-5, Pk01-10-9-4-7, Pk54-4-12-9, Pk20-2-1-9 and Pk13-1-1-9 and these were graded as resistant (R). Rest of the 12 pumpkin lines were free from infection with PRSV-W and graded as highly resistant (HR) (Table 2).

The percent disease index (PDI) value ranged from 13.3 to 58.3%. The highest PDI of was found on BARI mistikumra1 followed by Pk37-1-4-6. The lowest PDI was observed on Pk13-1-1-9 which was statistically similar to that of Pk20-2-1-9 and Pk02-2-1-6 (Table 2).

response-of-pumpkin-breeding-lines-to-papaya-ringspot-virus-w-3 response-of-pumpkin-breeding-lines-to-papaya-ringspot-virus-w-4

Relationship of plant growth and yield parameters with disease incidence and severity

The relationship of plant growth and yield parameters such as branches/plant, vine length, fruit number/plant, yield /plant TSS% with incidence of PRSV-W was linear and negative but not significant (Fig. 1 A, B and Fig. 2A, B & C).

The relationship of fruit number/plant, fruit yield/plant and %TSS of pumpkin with PRSV-W was liner and negative. The correlation coefficients of the relationship of fruit/plant (r=-0.019) and yield/plant (r=-0.067) with disease incidence was negative but not significant. The correlation co-efficient (r) was (- 0.019 and -0.067) and the contribution of regression (R2 =0.140 and R2 =0.175) was 14% and 17.55, respectively (Fig. 2A&B). The correlation co-efficient (r) was – 0.008 and the contribution of regression (R2 = 0.019) was only 19% (Fig. 2C). Virus in infected plant hampered the physiology and nutrient uptake of plants. So, yield and yield contributing characters are also affected. In virus-stressed plants, there was decreased yield in infected plant compared to healthy plant.

The symptoms recorded from pumpkin lines inoculated with PRSV-W are identical with the symptoms developed on other cucurbits due to viruses infection as reported by other investigators (Purcifull et al. 1984). Results of the present investigation reveal that disease incidence, severity, AUDPC and response of the tested pumpkin lines to PRSV-W are varied with the vitiations of the pumpkin lines. The variations may due to genetically variations in the pumpkin lines tested. Sherwood et al. (1986) also reported that differences in the pattern of incidence and degree of severity among cultivars may be due to variation in genetic make-up of the tested cultivars as well as the strain of the virus and possible co-infection with other viruses. Masud (1995) tested 27 pumpkin genotypes under field condition. Of them three were resistant and nine moderately resistant to pumpkin viruses. The resistance observed in those lines could be related to the existence of mechanisms that inhibit movement of virus from inoculated leaves to healthy leaves. Resistance could involve cellular membrane changes that impede the diffusion transport of infective virus particle from cell to cell, or an inhibition of virus particle replication in the leaf tissue of resistance plants (Gray et al. 1988).

Finding of the present experiment show that plant growth and yield of pumpkin are affected by the PRSV-W. The findings are consistent with findings of previous researchers (Fortun and Lopez 1982, Tattini et al. 1990, Huang 2003). They found that disease incidence was used and positive effects on plant growth could be proved. The positive effect of growth on the nutrients uptake was also proved with tomato, cucumber and other plants.


response-of-pumpkin-breeding-lines-to-papaya-ringspot-virus-w-7 response-of-pumpkin-breeding-lines-to-papaya-ringspot-virus-w-8

Finding of the present experiment show that plant growth and yield of pumpkin are affected by the PRSV-W. The findings are consistent with findings of previous researchers (Fortun and Lopez 1982, Tattini et al. 1990, Huang 2003). They found that disease incidence was used and positive effects on plant growth could be proved. The positive effect of growth on the nutrients uptake was also proved with tomato, cucumber and other plants.


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1Associate Professor, Dept. of Plant Pathology, Sher-e-Bangla Agricultural University, Dhaka-1207, 2PSO, Vegetable Division, HRC, Bangladesh Agriculture Research Institute, Gazipur-1701, 3Professor, Department of Plant Pathology, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur-1706
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