First report of Colletotrichum spp . causing diseases on Capsicum spp . in Sabah , Borneo , Malaysia

Blackish or orange liquid-like spots were found on (n=100) fruits of chillies (Capsicum) sold in five local markets in Kota Kinabalu, Sabah, Malaysia. Colletotrichum gloeosporioides and C. capsici were identified as the causal agents of an anthracnose disease. This is the first report of Colletotrichum spp. as the causal agent of anthracnose infected chillies in Sabah.


INTRODUCTION
The genus Capsicum comprises several species of chillies belonging to the family of Solanaceae.There are five important domesticated chilli species i.e. C. annuum, C. chinense, C. baccatum, C. frutescens and C. pubescens.This genus is native to the Americas and is now cultivated worldwide (Sanogo 2003).In Sabah, chillies are planted intensively at Kundasang, Keningau and many other areas.Chillies are important ingredients in local food and are used regularly.Anthracnose is a common disease attacking chillies in Sabah but the causal agents, the species of the genus Colletotrichum have have not been identified so far.According to Poonpolgul & Kumphai (2007), Thailand had encountered severe losses up to 80% due to a great fall in chilli production affected by Colletotrichum spp.whereas losses which were greater than 30% in plant production occurred in the United Stated due to anthracnose (Howard et al. 1992;Wilson et al. 1992).

MATERIAL AND METHODS
Sampling: Samples were collected from five local markets in Kota Kinabalu, Sabah, Malaysia, i.e.Central Market, Menggatal Market, Donggongon Market, Lido Market and Wong Kwok Market.
Fungi isolation: The samples were brought to laboratory in a sealed plastic bag.Lesions were excised, surface sterilized with 70% ethanol for 30s rinsed a few times with distilled water and dried on filter papers.They were cut into smaller pieces of approximately 5x5 mm size, cultured in petri dishes containing potato dextrose agar (PDA) and incubated at room temperature, in the dark, for 48 to 72 hr.
Morphological identification: An inoculum loop was used to scratch the mycelium of the colonies and mounted on the slides.Compound microscope and scanning electron microscope were used to observe the colony colour and form, appressorium features, shape, size and colour of spores from different isolates.Identification was made by comparing the characteristics of fungi following Charlie et al. (2001) and Deacon (1998).

RESULTS
One-hundred infected chillies with symptoms of anthracnose caused by Colletotrichum spp.were collected from the five local markets of Kundasang, Ranau areas, Papar and rural areas (Image 1).The lesions gradually turned darker, contained fluid as they matured.Lesions occured at different parts of the fruits as well as panicle in different sizes.
Out of 100 chilli samples collected, 50 isolates of Colletotrichum from the lesions, were two species i.e.Colletotrichum gloeosporioides and Colletotrichum capsici from five local markets in Sabah.Overall, the Central Market recorded the highest percentage of Colletotrichum spp.This was followed by the Wong Kwok Market and Donggongon Market which documented 24% and 20% of the total number of Colletotrichum isolates, whereas the least number of Colletotrichum isolates were from the Menggatal Market (Fig. 1).Totally 49 isolates of Colletotrichum gloeosporioides and one isolate of Colletotrichum capsici were successfully obtained.Colletotrichum gloeosporioides was mainly isolated from the Central Market followed by Wong Kwok Market.In contrast, this species was rarely found from the Menggatal Market (Table 1).Thus, the samples indicate infection mainly by Colletotrichum gloeosporioides and

Colletotrichum capsici.
Observations of morphological characteristics of the isolates showed similarities with published descriptions of Colletotrichum spp.(Cano et al. 2004).However in this study, Colletotrichum gloeosporioides strains indicated differences in growth patterns (Image 2).
The colour of conidia obtained varied between olive and white while the reverse colour for olive colonies varied from dark olive-grey to brown.For white-coloured colonies, the reverse colour was whitish with a little olive (Image 2B).The olive coloured colony produced abundant sporulation while the white possessed sparse mycelium.Setae developed a dense, cylindrical layer around the conidiamata of the olive-coloured colonies, which gradually turned into dark olive-grey, as maturation progressed.White colonies retained their colour

Colletotrichum capsici
The isolates showed grayish-olive colonies while the reverse color was darker olive (Image 4).Sporulation of this species was sparse and the acervuli were scattered.This species formed smooth, circular margin in the colony.The gray-whitish mycelium of Colletotrichum capsici gradually developed from the isolates from the second day of culture.Colour of the colonies of Colletotrichum capsici is similar to Colletotrichum gloeosporioides.However, both of these Colletotrichum species showed distinct difference in conidia and appresoria shapes.The conidia of Colletotrichum capsici were falcate in shape while the appresoria were round to ovate in shape.The spore measurement of this species varied between 13.21-16.21µm long and 1.79-3.28µm wide.The conidiophores of all the colonies branched below the surface of media as they developed.

DISCUSSION
In this study, the commonly available Capsicum annuum, C. baccatum and C. frutescens collected from the local markets in Kota Kinabalu, Sabah were studied.Occurrence of Colletotrichum spp.induced chilli anthracnose at the Central Market and Wong Kwok Market was higher when compared to the other three sites, most probably due to high humidity and wetness.This is consistent with the assumption of Arauz (2000) that excess moisture enables spores to be released from acervuli.On the other hand, the Menggatal Market and Lido Market smaller in size, and the stalls at Lido Market grouped into compartments according to types of goods sold decreased the percentage of humidity reduced the occurrence of chilli anthracnose.Arauz (2000) stated that 20-30 0 C range is the optimal condition for Colletotrichum gloeosporioides to form appresoria and

A B C D E F
germinate).In this study, Colletotrichum gloeosporioides was found to be the most dominant pathogen (98%), significantly more than Colletotrichum capsici , in agreement with the study of Ellett (1989) and Manandhar & Hartman (1995).This study reveals agreement with Black & Wang (1993-2002) that Colletotrichum gloeosporiodes and Colletotrichum capsici were mostly found in ripe chillies collected from Kota Kinabalu, Sabah.Prusky (1996) postulated that fruits are attacked by the pathogen early in their development.The fungi remain as germinated appressorium during quiescent period develop brown-black spots on the pericarp and soft rot in the mesocarp when fruit ripens for harvest.Infection of chillies by Colletotrichum spp. is mainly due to ample nutrients and limited antifungal compounds formed during fruit ripening (Prusky 1996).

CONCLUSION
The two species of Colletotrichum spp., i.e.Colletotrichum gleoesporioides and Colletotrichum capsici were isolated from the chilli lesions collected from local markets in Kota Kinabalu, Sabah.Although Colletotrichum acutatum is reported as one of the common pathogens on chilli lesions, it was not found in this study.The conidia of Colletotrichum gloeosporioides were straight with obtuse apex, hyaline, cylindrical to clavate, and sometimes fusiform like Colletotrichum acutatum.Appresoria were mainly present in mature colonies.They were clavate and lobed.The spore measurement for this species ranged within 3.93-12.14µm long and 1.43-2.14µm wide.Isolates from Colletotrichum capsici, had similar olive color of Colletotrichum gloeosporioides,.However, they possessed falcate conidia distinct from Colletotrichum gloeosporioides.In addition, Colletotrichum capsici formed round to ovate appresoria.The spore measurement of this species was 13.21-16.21µm length and 1.79-3.28µm width.Colletotrichum gloeosporioides and Colletotrichum capsici grew better on Potato Dextrose Agar (PDA) than in Malt Extract Agar (MEA) and Yeast Extract Agar (YEA).Growth rates of Colletotrichum spp.were higher under light exposure than in dark.The samples are stored as stock culture in the Institute for Tropical Biology and Conservation (ITBC), Universiti Malaysia Sabah.