African Crop Science Journal, Vol. 11. No. 1, 2003, pp. 49-53

SHORT COMMUNICATION

EVALUATION  OF HOST-PATHOGEN INTERACTION OF MYCOVELLOSIELLA CAJAN WITH PIGEONPEA

F.M. GATHECA and A.W. MWANG'OMBE¹

National Museums of Kenya Molecular Genetics Department, P. O. Box 40658, Nairobi, Kenya
¹Department of Crop Protection, University of Nairobi, P. O. Box 30197, Nairobi, Kenya

Received 11 October, 2001;
Accepted 17 December, 2002)

Code Number: cs03007

ABSTRACT

Although  Mycovellosiella cajan  is an important pathogen limiting production of pigeonpea through transmission of Mycovellosiella leafspot, little is known about the structural formation of either the host (pigeonpea) or the fungus.  Thus, this study was conducted to assess any differences in structure as an indication of resistance in pigeonpea genotypes to Mycovellosiella cajan. Germtube growth was not different in either of the pigeonpea germplasms assessed.  Penetration in pigeonpea by Mycovellosiella cajan was inhibited by production of stomatal exudates in resistant varieties.  In susceptible varieties germtube grew towards the stomata and penetrated either directly or through the stomata.  In resistant varieties germtube bypassed the stomata and the point of penetration was not easily noticed.  Mycovellosiella cajan sporulated in susceptible pigeonpea leaves 16 - 18 days after inoculation and in resistant varieties, conidiophores were not observed even after 16 days.  In susceptible varieties there was rupturing of the stomata due to mass production of conidiophores through the stomata.

Key Words: Cajanus cajan, germplasm, leafspots

RÉSUMÉ Malgré que les  Mycovellosiella cajan est un important pathogène  limitant la production de pigeonpea à travers la transmission de tâches de Mycovellosiella cajan sur les feuilles, il y a une connaissance limitée sur la formation structurale de l'hôte (pigeonpea) ou du fongi . cette étude était conduite pour évaluer toutes les différences dans la structure comme indication de la résistance dans les génotypes du pigeonpea à la Mycovellosiella cajan. La croissance des tubes de germes n'était pas différente dans le pigeonpea et les germplasmes étudiés. La pénétration du Mycovellosiella cajan était par la production de stomatal exudates dans les variétés résistantes. Dans les variétés susceptibles, les tubes des germes depassant le stomata et le point de pénétration étaient facilement observés. Mycovellosiella cajan produit dans les feuilles de pigeonpea susceptibles 16 à 18 jours après l'inoculation et dans les variétés résistantes, les conidiophores n'étaient pas observés même après 16 jours. Dans les variétes susceptibles, il y avait une rupture du stomata à cause de la production de conidiophores à travers le stomata.

Mots Clés: Cajanus cajan, germplasme, mildiou

INTRODUCTION

Mycovellosiella cajan (Henn) Rangel ex trotter (syn. Cercospora cajan Henn Vellosiella cajan (Rangel) causes a foliar disease of pigeonpea commonly found in areas with high rainfall or during wet seasons in drier areas where pigeonpea are grown (Khan and Rachie, 1972).  Mycovellosiella leafspot disease has been reported as one of the most important diseases of pigeonpea in Africa, North and South America and Asia.  Infection of a susceptible host results in ashy grey leafspot symptoms.  The disease also infects stems and pods.  The foliar symptoms typically develop later in the growth of the crop often after flowering as reported for the leafspot of cowpea (Nakawuka and Adipala, 1999).  Mycovellosiella leafspot can reduce pigeonpea yield by upto 80% (Njoya, 1991).

The initial interaction between plant pathogenic fungi and their hosts involve physiological changes in both organisms that, in the fungus, reflect adaptations necessary for establishment of parasitic relationship (Hood and Shew, 1997).  Generally, no appressoria are formed prior to penetration but in some Cercospora species, appressoria formation has been observed on inoculated leaf surface of some plant such as sugarbeet (Solel and Minz, 1971).

Sporulation of Cercospora species on host surface usually occurs within 21 days following inoculation and is dependent on light, host susceptibility and site of infection (Beger and Hanson, 1963). During sporulation conidiophores arise from beneath the epidermis in the substomatal chamber or between the guard cells.  As the conidiophores enlarge, guard cells are forced apart as this usually result into ruptured epidermis.

The objective of this study was to identify any difference in structure formation of either the host (pigeonpea) or the fungus (Mycovellosiella cajan) as an indication of resistance in pigeonpea genotypes to Mycovellosiella cajan.

MATERIALS AND METHODS

Host and pathogen maintenance.  Histo-pathological studies were conducted using nine pigeonpea cultivars, three of which are susceptible (MKS KO 161/1, MKS KO 115 and MKS KO 252), three intermediate resistant (NPP 670, MKN KO 74 and ICPL 93015) and three resistant (KZ 56, ICPL 87109 and ICEAP 00554) to Mycovellosiella cajan.  The seeds were surface sterilised using 10% sodium hypochlorite and rinsed using 5 changes of sterile distilled water to eliminate seedborne inocular prior to planting.  Treated seeds were sown in steam sterilised forest soil, animal (cowdung) manure and some gravel mixed in ratio of 10:1:1 by volume.  Nyabundi (1980) reported good growth of pigeonpea in this soil composition.

The isolate of Mycovellosiella cajan used throughout this study was obtained from diseased leaves of NPP670, a pigeonpea breeder's line susceptible to Mycovellosiella cajan.  It was morphologically typical of isolates of Mycovellosiella cajan collected from pigeonpea landlaces from farmer's field at Taita Taveta, Embu, Nyambene, Kilifi and Malindi districts in Kenya.  It was also typical to other breeders line isolates in pathogenicity tests conducted under greenhouse conditions.  Cultures were maintained and inoculum produced on pigeonpea leaf decoction agar (300g of freshly picked Cajanus cajan leaves and 20g-agar/litter of sterilised distilled water autoclaved at 121°C for 15 minutes).  Approximately, 20 ml of the solution was dispensed into each of the sterile petri dishes after cooling to 45 - 48°C in a water bath.  Inoculum were harvested by removing 3 to 4 weeks old colonies of Mycovellosiella cajan from cultural plates by suspension in sterile distilled water.  The resulting suspension was filtered twice through four layers of cheesecloth to remove agar and hyphae.  The concentration of conidia was determined by haemocytometer and diluted with sterile distilled water to 2 x 10⁶ conidia per ml.

Inoculation of test plants was done 120 days after planting.  Plants were inoculated using a modified inoculation technique of Van der Vossen et al. (1976). A 2 x 10⁶ conidial/ml suspension of Mycovellosiella cajan was applied on both sides of all the leaves present on the plants using a half litre Baygon atomiser (Bayer East Africa Ltd.) held at a distance of 10 - 15 cm away until runoff.  A double inoculation after 48 hour interval was applied.

Scanning electron microscopy, leaf tissue discs were cut out using a 1 cm diameter cork borer after 6, 12, 24 and 48 hours after inoculation and thereafter on a daily basis until the 30^th day. Tissue fixation was done according to the method described by O'Connell et al. (1984). The inoculated tissues were immersed in 2.5% (w/v) glutaraldehyde fixative buffered in 0.05 M potassium phosphate (pH 8.0) in stoppered glass vials.  The tissues were left in the fixative for 4 hours at 4°C after which they were thoroughly washed 3 times for 30 minutes each in the same buffer.  The samples were post fixed in 2% Osmium tetroxide buffered in 0.05 M potassium phosphate pH 6.8 for 6 hours at 4°C. The specimens were later washed thoroughly in the same buffer, 3 times for 30 minutes each.  The specimen were dehydrated in a graded series of ethanol (50%, 70%, 80%, 90%, 100%) at 10 minutes intervals and dried in a CPD 030 critical point drier.  The specimens were mounted on the metal stubs and gold coated using ion sputter JFC-1100 (JEOL LTD. Japan).  The specimen were viewed under JOEL JSM- T330A scanning electron microscope and observations were made on conidia germination, penetration and sporulation of Mycovellosiella cajan on pigeonpea leaves.

RESULTS

Germination was noted as early as one and half hours after inoculation.  Conidia germination peaked up considerably 3 hours after inoculation in susceptible genotypes and germtubes were produced at either one or both tips of the conidia while others emerged from the sides.  However, for the  resistant  and intermediate resistant varieties  conidia germination was observed 3 hours after inoculation and although germtube growth was rapid, few stomatal ingress was observed instead growing germtube passed immediately beside or across the stomata (Fig. 1A) and the point of penetration was not easily noticed.  In susceptible genotypes, germtube growth was rapid and side branches were formed near the stomata and grew either directly or through the stomata opening (Fig. 1B).  Penetration in resistant varieties was inhibited by production of stomatal exudates (Fig. 1C).  In resistant varieties most of the conidia on the leaf surface seemed deformed and most of the stomata were closed (Fig. 1D).  Conidiophores were produced about 16-20 days after inoculation in susceptible cultivars (Fig. 1E).  Conidiophores bearing abundant conidia caused rupturing of the stomata and were observed 20 days after inoculation in susceptible varieties (Fig. 1F) and this was not observed in resistant varieties even after 30 days after inoculation.

DISCUSSION

Scanning electron microscopy study revealed that some resistant varieties produced stomatal exudates that inhibited fungal penetration.  Marschner (1995) reported that in the epidermal cells of barley, more than 90% of the soluble carbohydrates are β-cyanoglucosides which are likely to be of particular importance in resistance against powdery mildew.  The rate and time of sporulation was found to be an important distinguishing feature in the reactions of the categories (resistant, intermediate resistant, and susceptible) of pigeonpea cultivars to Mycovellosiella cajan. The initial stagnation of events and the suppression of sporulation on the resistant cultivars noted here is a feature also reported in other host-pathogen relations, for example, resistance of coffee arabica to Colletotricum coffeanum Noack (Gichuru et al., 1996).  This may be as a result of some physical or chemical barriers.

ACKNOWLEDGEMENTS

Thanks are due to Belgium Administration Development and Co-operation (BADC) for financing this work and Drs. Rose Njeru and Clive Wells for their review comments.

REFERENCES