PESTALOTIOPSIS GUEPINII (DESM.) STAY. – A NEW PATHOGEN OF BLACK SPOT DISEASE OF ROSE IN BANGLADESH
Shamim Shamsi and Anita Ghosh. 2013. Pestalotiopsis guepinii (desm.) Stay. – a new pathogen of black spot disease of rose in Bangladesh. Bangladesh J. Plant Pathol. 29 (1&2): 11-14.
An investigation was conducted during November 2009 to October 2010 to determine causal fungi of black spot disease of rose (Rosa centifolia L.). Black spot infected leaf samples were collected from different locations of Dhaka city. The fungi associated with the samples were isolated and identified. The principal fungal pathogen associated with the diseased specimens was Diplocarpon rosae Wolf (imperfect stage Marssonina rosae). Other fungal pathogens associated with the disease were Pestalotiopsis guepinii and its two culture types (P. guepinii-1, P. guepinii-2). The fungi belong to the class Coelomyecetes under the Division Deuteromycita. The fungi were frequently isolated from black spot infected leaf samples of the rose. After inoculation of detached leaves and seedlings of rose, P. guepinii and its two culture types developed characteristics symptoms of black spot. The pathogen was reisolated from black spot infected inoculated leaves to fulfill Koch’s postulate. The findings of the investigation indicate that P. guepinii is another fungal pathogen of black spot of rose in addition to D. rosae. This is a new record about causal agents of black spot disease of the cut flower.
Rose (Rosa centifolia L.) is grown throughout the world for their beautiful flower and fragrance. The ornamental plant is also grown in Bangladesh. Diplocarpon rosae Wolf and its imperfect stage Marssonina rosae (Lib.) Died is well documented pathogen of black spot of rose. Black spots are circular with a perforated edge and reach a diameter of 14 mm. Severely affected plants however, do not show the circular spots as they coalesced together and form large lesion (Debner 1988). From India, Mukerji and Bhasin (1986) reported leaf spot of rose caused by Pestalotiopsis versicolor. Islam et al. (2010) reported seven diseases of rose from Bangladesh. The diseases, in order of their prevalence, are Botrytis blight (Botrytis cinerea), Cercospora leaf spot (Cercospora puderi), rose mosaic (Rose Mosaic Virus), black spot (Diplocarpon rosae), die-back (Botryodiplodia theobromae), Alternaria leaf spot (Alternaria alternata) and stem canker (Crytosporella umbrina (Speg.) Stey. In Bangladesh, reports on association of fungal pathogens with black spot symptoms are not available. The present investigation was undertaken to identify the fungal pathogens associated with black spot disease of rose plant other than D. rosae.
MATERIALS AND METHODS
Healthy and diseased leaves of rose were collected from different locations of Dhaka city during November 2009 to October 2010. Fungi were isolated from black spot infected leaf samples following “Tissue Planting method” on PDA medium and pure culture of the isolated fungi were prepared following single spore method (Tuite 1969). Morphological characteristics of the fungi were recorded under a compound microscope and identified using standard key books (Barnett and Hunter 2000, Booth 1971, Ellis 1971, 1976, Sutton 1980, Ellis and Ellis 1997). All diseased specimens and associated fungi were preserved in the Herbarium of the Department of Mycology and Plant Pathology, University of Dhaka.
Pathogenicity test of the isolated fungi was performed following modified detached leaf assay technique (Azad and Shamsi 2011). Fresh, healthy and mature leaves of rose were collected, washed with distilled water, surface sterilized with 1.0% Clorox for five minutes and rinsed in sterilized water. Ventral and dorsal sides of the leaflets with and without pricking with needles were inoculated with 2 mm diameter mycelial block of the isolated fungi previously grown on PDA medium for seven days. Another set of leaves with and without pricking and without inoculation were maintained, which served as controls. Three replicated leaflets were used for each treatment. The inoculated leaflets were placed in Petri dishes containing water soaked filter paper and cotton ball to maintain sufficient humidity to initiate infection. The plates were incubated at 25-28C. The inoculated and non inoculated leaflets were checked for symptom development starting from 3 days of inoculation and continued up to 7-10 days.
Seedling inoculation method was also followed to confirm pathogenicity of the isolated fungi. Healthy seedling of rose plant was transplanted in pots (30 cm Diam.) containing sterilized soil at three seedlings per pot and allow to grow for three month in a nethouse providing necessary water and nutrients. Healthy leaves of seedlings were washed with sterilized water and surface sterilized with 1.0% Chlorox and rinsed with sterilized distilled water.
Surface sterilized leaves were pricked with sterilized needle. Pricked and unpricked leaves were inoculated with the test fungi by rubbing sporulating PDA culture of the test fungus. Leaves under control received only fresh PDA medium without fungal inoculum. Plants were covered with polythene bags to maintain proper humidity level and to avoid contamination. Three seedlings were inoculated for each treatment. The inoculated plants were placed in a clean bench. The plants were examined daily and continued up to 10 days to record the development of symptoms. Symptoms developed on artificial inoculated leaves were recorded and compared with the symptoms of those observed on naturally inoculated leaves. The fungus was resonated from the inoculated leaves of rose to fulfill Koch’s postulates.
RESULTS AND DISCUSSION
Marssonina rosae was isolated from black spot infected leaves of rose. In addition to M. rosae, another fungal pathogen, P. guepinii and its two culture types (P. guepinii-1, P. guepinii-2) were frequently isolated from rose leaves having black spot symptoms (Plate I A, B and C). Their cultural characteristics are summarized in Table 1. Other fungi associated with black spot infected leaves of rose were Cladosporium cladosporioides (Fresen.) de Vries, C. oxysporum Berk. & Curt. and a species of Penicillium.
Taxonomic enumeration of Pestalotiopsis guepinii
Colonies on PDA having pH 6.0 and incubated temperature 25±1C are white, cottony. Hyphae hyaline, septate and profusely branched. Acervuli are black, shinning, conspicuous, conidiomata 200 µm long. Conidiophores short hyaline, 10-15 µm long and 1-2 µm wide, mostly aseptate, with 1-2 proliferation. Conidia blackish brown, mostly three septate with 2-5 (mostly 3) hyaline appendages at the apex and a short hyaline appendages at the base, apical appendages 16.5 – 26.1 µm long and a basal 4.2 – 7.8 µm long (Plates I and II). Based on these morphological characteristics, the fungus was identified as Pestalotiopsis guepinii (Desm.) Stay. (Sutton 1980).
Characteristic symptoms of black spot developed on rose leaves which were pricked dorsally and inoculated with Pestalotiopsis guepinii as dark brown lesion. The fungus was reisolated from inoculated rose leaves showing characteristic symptoms to fulfill the Koch’s postulates. Two culture types of P. guepinii (1&2) also produced characteristic symptoms of black spot on inoculated rose leaves. Pestalotiopsis guepinii produced 8-10 mm dark lesion around the point of inoculation. Subsequently P. guepinii-1 produced 10-11 mm and P. guepinii-2 produced 8-12 mm irregular dark lesion around the point of inoculation. Control leaves did not show any symptoms of disease (Plate III).
Results of the present study reveal that P. guepinii and its two culture types can develop symptoms of black spot only when test leaves are pricked dorsally before inoculation. It indicates that injury is essential for penetration of the fungus. Black spot caused by D. rosae and its imperfect stage has been reported earlier from Bangladesh (Islam et al. 2010) as well as abroad (Willium 1949, Debner 1988, Mukerji and Bhasin 1986). Reports on the association of P. guepinii with black spot infected rose leaves under natural conditions and its pathogenicity to cause the disease on inoculated rose leaves are not available in Bangladesh or abroad. The findings indicate that P. guepinii is another pathogen of black spot of rose and this is a new information on causal agent of the disease.
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