Annals of the Sri Lanka Department of Agriculture. 2008.10:129-136.
DEVELOPMENT OF VARIETY SCREENING METHOD FOR ANTHRACNOSE DISEASE OF CAPSICUM (Capsicum annum L.) R.G.A.S. RAJAPAKSE, W.A.R.T. WICKRAMAARCHCHE, S.M.I.S.K. SAKALASOORIYA, R.D.S.S. WIJESEKARA and J. KAHAWATTA Horticultural Crops Research and Development Institute, Gannoruwa, Peradeniya
ABSTRACT Anthracnose disease significantly affects the yield and quality of capsicum (Capsicum annum L.). Some capsicum varieties are tolerant to anthracnose but there are no reliable methods to identify resistant varieties. Therefore, experiments were conducted to develop a suitable screening method to identify anthracnose resistant varieties of capsicum under laboratory conditions. The predominant anthracnose pathogen found in the Central Province of Sri Lanka was Colletotrichum gloeosporioides. Colletotrichum capsici was rarely found. Conidia of Colletotrichum gloeosporioides germinated and produced appressoria within a few hours on fruit exudates of all tested varieties. Percentage of conidia germination of Colletotrichum gloeosporioides in fruit exudates of different varieties showed a significant correlation with the size of anthracnose lesions developed on fruits of each capsicum variety. The highest stimulation of conidia differentiation of pathogen was observed in fruit exudates of the variety â€œHungarian Yellow Waxâ€? which is highly susceptible to anthracnose disease. Chemical factors found in exudate obtained from fruits were separated into two fractions that are soluble in ether and in water. Chemical factors found in both ether and water soluble fractions enhanced conidia differentiation. Results revealed that stimulatory effect of exudates on conidia germination could be used to compare susceptibility or resistance against anthracnose among the capsicum germplasm as an in vitro test. KEYWORDS: Anthracnose, Appressoria, Capsicum, Colletotrichum.
INTRODUCTION Anthracnose disease is one of the major constraints to the profitable cultivation of capsicum during rainy periods. Recently, a serious incidence of anthracnose of capsicum was observed in many areas of Sri Lanka. Losses occurred in the field and during transit. In addition to direct losses the disease impairs quality so that even saleable fruit commands a lower price than would be obtained in the absence of the disease. It has been reported that apart from the pre-harvest losses, pod quality deterioration of capsicum due to anthracnose ranges from 20 - 50% in Sri Lanka. However, estimates of losses are very high and varied with climatic conditions of the growing season (Kim and Park 1988). Anthracnose of capsicum, in common with similar disease of other crops, is caused by species of the Genus Colletotrichum (Agrios, 1988). Characteristically, it is predominantly associated with lesions on matured fruit,
130 RAJAPAKSE et al.
but also causes the die-back of stems and branches in capsicum (Abeygunawardhna, 1969). Several species of Colletotrichum, C.capsici (Sydow) Butl. and Bisby, C. gloeosporioides (Pent.) Penz. and Sacc., C. acutatum Simmonds, C.coccodes (Wallr.) Hughes, C.graminicola (Ces.) Wils., have been implicated in the anthracnose disease of chilli in various part of the world (Hadden and Black, 1988). Two species of Colletotrichum i.e. C. capsici and C. gloeosporioides have been identified as causal agent of anthracnose disease of chilli in Sri Lanka (Rajapakse, 1998). To date, research on the anthracnose disease in Sri Lanka has been mainly directed to the development of chemical control methods and also to identify resistant varieties. From this research, a number of control measures have been devised and recommended to farmers. These measures included the use of systemic and contact fungicides as seed dressings and foliar sprays. However, they have not provided a satisfactory level of control, especially in wet conditions. The most economical way to minimize crop losses due to disease is cultivation of resistant varieties. However, there is no reliable method to screen anthracnose resistant varieties of capsicum. At present, resistance of capsicum varieties against anthracnose pathogen is measured under natural infections or artificial spraying of inoculum suspension onto the pods without considering mode of anthracnose development on pod surfaces. Therefore, development of an effective variety screening method to identify anthracnose resistant chilli varieties is an important research requirement to minimize crop losses. MATERIALS AND METHODS Pathogen isolation and identification Anthracnose affected capsicum fruits were collected from farmer fields of different locations in the Central Province. Pathogens were isolated from anthracnose lesions of disease-affected fruits on Potato Dextrose Agar (PDA). Single spore isolates of pathogen were prepared from mycelia of single conidia cultures grown on PDA. Pathogens were identified on the basis of size and morphology of sporulating acervuli, conidia and morphology on culture medium by microscopic observations. Isolates of the pathogen were stored in PDA slants for further studies. Pathogenicity of all isolates was tested by wound inoculation with conidia suspension i.e pin prick method and subsequent anthracnose development on fruit surfaces (Rajapakse, 1999).
VARIETY SCREENING FOR ANTHRACNOSE ON CAPSICUM 131
Collection of exudates from capsicum fruits Fruits for this experiment were collected from the field grown plants of the eight capsicum varieties [HCA 1, HCA 2, HCA 3, Hungarian Yellow Wax (HYW), CA 8. Matale selection (MS), Shivani and Hacona]. Fully grown, green mature stage fruits of capsicum were obtained from healthy plants. Fruits were carefully detached from plants and washed with Sterile Distilled Water (SDW) and then wiped with cotton wool soaked in 90% ethanol to reduce microbes on pod surface. Five SDW drops of 10 µl volume were separately placed using a micropipette on surface of each fruit of all tested varieties and left for 16 hr in a moist chamber. Total of 6 fruits from each 6 plant were used for collection of exudates from each variety. To avoid microbial activity, exudates were then stored at -20°C until later analysis. Fractionation of exudates Compounds in exudates were separated into ether soluble and insoluble fractions. Exudates (5 ml) were shaken with 15 ml diethylether in a separating funnel. The ether fraction was then separated from the water fraction. Process was repeated five times and the ether washings were bulked and dried over anhydrous Na2SO4. The ether and water-soluble fractions were evaporated to dryness using a vacuum evaporator in a water bath at 38°C and finally the solutions of ether soluble and insoluble compounds were obtained by adding 5 ml of SDW. Effect of ether soluble and water soluble fractions of exudates on conidia germination and appressoria formation Bioassay Conidia germination, appressoria formation and anthracnose development of pathogen in fruit exudates of eight varieties were determined. Isolate of C. gloeosporioides from capsicum fruits were used for all inoculation and bioassay experiments. Conidia for all experiments were obtained from 14 days old cultures made on Potato Dextrose Agar (PDA) at room temperature (28°C– 30°C). Conidia were harvested by adding 10 ml SDW to the culture dishes, which were then gently shaken. The suspension was then transferred into presterilized centrifuge tubes, which were then spun at 5000 rpm for 4 min. The supernatant was discarded and the conidia contained pellets were resuspended in fresh SDW. This process was repeated four times. Density of conidia in the final suspension was measured using a haemocytometer and adjusted to the 5x105 conidia /ml with SDW (Rajapakse, 1998). Ten microlitre drops of conidia suspension of both isolates were wound inoculated on three sites of fruit surfaces to observe anthracnose lesion development on fruit surfaces.
132 RAJAPAKSE et al.
Then fruits were transferred into humid plastic boxes lined with moisture paper pre-soaked in SDW. The experiment involved a Completely Randomised Block Design with three replicates. To determine the effect of ether soluble and water soluble fractions of fruit exudates on germination and appressoria formation by conidia, bioassays were conducted on glass slides. A suspension of conidia in SDW was mixed with equal volumes of the exudates or ether soluble or water soluble solutions and 10 µl drops were placed on the clean glass slides. The slides were then placed in sealed chambers, lined with moist filter paper to prevent evaporation, and incubated for 16 hrs at 28 °C. Slides were removed briefly, dried by placing them in an oven at 50°C and then a drop of lactophenol containing trypan blue (0.03%) was added. Percentage germination and the percentage of germinated conidia with appressoria were assessed by microscopic observations. The experiments were repeated twice with three replicates on each occasion. Values were based on count of 200 conidia in all replicates. The data were transformed to arc sin and analysed by ANOVA using a statistical programme (MSTATC). RESULTS AND DISCUSSION Isolates of anthracnose pathogen of capsicum were identified by comparison of their colony morphology on PDA with published data. According to the colony morphology on PDA medium and the shape and size of conidia, isolates were identified as C. capsici and C. gloeosporioides (Table 1). Pring et al. (1993) and Sutton (1992) showed that variation between isolates is typical for many species in the Genus Colletotrichum. However, characteristics of tested isolates were within the published range of C. capsici and C. gloeosporioides (Sutton, 1992). Isolates tested for their ability to induce lesions in mature fruits using the pin prick method indicated that tested isolate of C. capsici and C. gloeosporioides had the ability to develop anthracnose lesions on capsicum fruits. The disease was identified by large 1 – 1.5 cm diameter, brown, circular depressions and the fungus appeared as brown acervuli on the surface of inoculated capsicum fruits. Most of the diseased capsicum fruits collected in this study were found to be infected with C. gloeosporioides and a few fruits were found with C. capsici infection. The predominant pathogen of anthracnose disease in the central region of Sri Lanka was C. gloeosporioides. Therefore, studies reported in this paper were conducted with isolate of major pathogen i. e. C. gloeosporioides. Results of germination assay (Table 2 and 3) show that there were significant differences in conidia germination and appressoria formation of C. gloeosporioides in exudates of different capsicum varieties. However, the highest number of conidia germinated and formed appressoria in exudates
VARIETY SCREENING FOR ANTHRACNOSE ON CAPSICUM 133
of fruits of the variety HYW. The lowest conidia germination and appressoria formation were observed in fruit exudates of the variety Hacona. Table 1. Characters of fungal isolates of C. gloeosporioides and C. capsici collected from anthracnose affected capsicum pods. Characters of fungi isolates
Morphology and culture characters of isolates C. capsici C. gloeosporioides
Colony colour on PDA
White initially then turned light brown or brown, colony margin smooth Dark brown
Grey initially then turned light brown or brown, colony margin wavy Brown
Black colour masses, conidia present inside. Black colour, straight, septate, 60192 µm in length and 4-6 µm in width
Brown colour present inside. Absent
Sickle shaped, aseptate, vacule present in centre, 16-32 µm in length and 2-4 µm in width, Conidia germinate and produce appressoria at the end of germ-tube on plant surfaces, but rarely in distilled water
Cylindrical with round end shaped, aseptate, Fat globules present inside, 12-18 µm in length and 2-4 µm in width, Conidia germinate and produce appressoria at the end of germ-tube
Appressoria on fruit surface
Oval shape, abundant and black colour
Oval shape, abundant and black colour
Reverse colony colour on PDA Acervuli Setae
The original exudates and their water and ether fractions stimulated the germination of conidia relative to the water controls (Table 2 and 3). It was revealed that materials responsible for conidia germination and appressorium formation are either ether or water soluble. Conidia differentiation was very low in SDW on glass slides. Similar observations were made by Swinburne (1976) with Colletotrichum musae on banana, which was ascribed to the presence of solutes leaching into the inoculum drop from the host cells. Rajapakse (1999) showed that conidia of C. capsici germinated and formed appressoria to varied degrees in fruit exudates from different chilli varieties. The rate of leaching of solutes from plant cells is related to the integrity of the plasma membrane (Tukey, 1970). Rajapakse (1998) showed that chemical factors such as phenylalanine, found in fruit exudates of chilli were stimulatory to conidia differentiation of C. capsici. The lesion development was assessed as length of the lesion after 9 days of inoculation. Significant differences in lesion development were found between the fruits of different varieties (Table 4). Highest lesion development was observed on fruits of variety HYW and lowest was observed on fruits of variety Hacona (Table 4). Percentage germination of C. gloeosporioides in fruit exudates of
134 RAJAPAKSE et al.
the capsicum varieties had a significant correlation with the conidia size of the anthracnose lesion on fruits (r2 = 0.81) (Fig. 1). Therefore, stimulatory effects of exudates of different varieties of conidia germination could be used to compare anthracnose susceptibility/resistance among the capsicum germplasm as an in vitro test. Table 2. Varieties
The effect of exudates obtained from fruits of capsicum varieties on the germination of conidia of C. gloeosporioides at 16 h incubation. Percentage conidia germination of C. gloeosporioides** *EX EF WF
HCA 1 28.6 ab 12.7 b 10.0 b abc b HCA 2 27.8 12.3 10.2 b ab bc HCA 3 28.4 10.9 7.4 c a a HYW 33.3 24.4 13.2 a cd b CA 8 29.0 12.4 7.1 c bcd d MS 24.8 7.9 7.0 c bcd cd Shivani 21.6 8.1 5.9 cd d dc Hacona 18.6 6.2 5.9 cd Control (SDW) 2.9 e 4.2 c 3.5 d * EX – Fruit exudates, WF – Water fraction of fruit exudates, EF – Ether fraction of fruit exudates, SDW – Sterile distilled water, ** mean of replicates In each column, values followed by the same letter are not significantly different, following DMRT at p=0.05. Table 3.
The effect of exudates obtained from fruits of capsicum varieties on the appressoria formation from germinated conidia of C. gloeosporioides at 16 h incubation. Percentage appressoria formation of C. gloeosporioides** *EX EF WF
HCA 1 21.2 b 19.0 a 11.9 eb cd eb HCA 2 13.3 15.4 9.7 ebc bc eb HCA 3 17.4 13.3 9.2 ebc a b HYW 25.4 16.6 11.9 eb bcd ebc CA 8 15.6 10.0 14.3 e MS 14.0 cd 13.1 eb 11.7 eb Shivani 10.5 de 9.7 bc 12.7 eb de ab Hacona 9.2 12.0 6.5 bc e c Control (SDW) 3.6 3.6 3.5 c * EX – Fruit exudates, WF – Water fraction of fruit exudates, EF – Ether fraction of fruit exudates, SDW – Sterile distilled water, ** mean of replicates In each column, values followed by the same letter are not significantly different, following DMRT at p=0.05.
VARIETY SCREENING FOR ANTHRACNOSE ON CAPSICUM 135 Table 4. Lesion size of anthracnose on fruits of capsicum varieties. Varieties
Lesion size (cm)
HCA 1 2.7 b HCA 2 2.8 b HCA 3 2.6 bc HYW 3.2 a CA 8 2.3 d MS 2.6 bc Shivani 2.4 cd Hacona 2.1 d Values followed by the same letter are not significantly different, following DMRT at p=0.05. 40
y = 13.355x - 9.9556 R2 = 0.8126
Conidia germination (%)
35 30 25 20 15 10 5 0 1.5
Lesion size on fruits (cm)
Figure 1. Relationship between conidia germination (%) in fruit exudates and size of the anthracnose lesion on fruits of capsicum varieties.
CONCLUSIONS The predominant pathogen of anthracnose disease of capsicum in the Central Province of Sri Lanka is Colletotrichum gloeosporioides. Stimulatory compounds, which are responsible for conidia germination and appressoria formation, are present in fruit exudates of capsicum. Highest stimulation of conidia differentiation of the pathogen was observed in fruit exudates of the variety Hungarian Yellow Wax which is highly susceptible to anthracnose disease. Rate of conidia differentiation of C. gloeosporioides by exudates could be used to compare susceptibility/resistance among the capsicum germplasm as an in vitro test to anthracnose. REFERENCES
136 RAJAPAKSE et al.
Abeygunawardhana, V.W. 1969. Diseases of cultivated plants. Colombo Apothecaries LTD, Colombo. 167-169 p. Agrios, G.N. 1988. Plant pathology, 3rd edition, Academic Press, London, UK. 324-331 p. Hadden, J.F. and I.L. Black. 1988. Anthracnose of pepper caused by Colletotrichum spp: Tomato and pepper production in the tropics. In Proceedings of the International Symposium of Integrated Management Practices. Pp 317-319. AVRDC, Taiwan. Kim, C.H. and K.S. Park. 1988. A predictive model of disease progression of red pepper anthracnose. Korian Journal of Plant Pathology 4:325-331. Pring, R.J., C. Nash and J.A. Baily. 1993. Infection process and host range of Colletotrichum capsici. Physiological and Molecular Plant Pathology 46:132-152. Rajapakse, R.G.A.S. 1998. Some observations of anthracnose disease caused by Colletotrichum species in Sri Lanka. PhD thesis, University of London, UK. Rajapakse, R.G.A.S. 1999. Mode of anthracnose development in chilli (Capsicum annuum L.) pods. Annals of the Sri Lanka Department of Agriculture 1:247-266. Sutton, B.C. 1992. The Genus Glomerella and its anamorph Colletotrichum. Collototrichum biology, pathology and control. CAB international, UK. 1-26 p. Swinburne, T.R. 1976. Stimulation of germination and appressoria formation by Colletotrichum musae (Berk. and Curt.) Arx. in banana leachete. Phytopathologische Zeitschrift 87:74-90. Tukey, H.B. 1970. The leaching of substances from plants. Annual Reviews of Plant Physiology 21:305 -324.