African Crop Science Journal African Crop Science Society ISSN: 1021-9730 EISSN: 2072-6589 Vol. 7, Num. 4, 1999, pp. 523-530

African Crop Science Journal, Vol. 7. No. 4,  pp. 523-530, 1999                                                              

Colonisation of resistant and susceptible bean tissues by Phaeoisariopsis griseola (Sacc) Ferr

I. N. Wagara, A. W. Mwang’ombe and  G. M. Siboe
Department of Crop Protection, University of Nairobi, P.O. Box 30197, Nairobi, Kenya

Code Number: CS99043

ABSTRACT

Fungal development and the associated cellular reactions in three bean (Phaseolus vulgaris L.) cultivars/lines inoculated with Phaeoisariopsis griseola isolate PG18 were studied. Differences in the extent of fungal development and host cell integrity were used to compare the reactions of the  three cultivars/lines (resistant, intermediate resistant and susceptible). Conidia of P. griseola germinated 4 hours after inoculation by producing germ tubes at either one, both tips or at the sides. However, germination in the resistant bean line M26 was slightly inhibited and most of the conidia had not germinated 4 hours after inoculation. Penetration occurred 24 hours after inoculation and was either direct or through the stomata. On  M26, minute brown flecks were observed on the inoculated area 4 days after inoculation, whereas in cv Rosecoco-GLP-2 (susceptible) and line M29 (intermediate resistant), development of conspicously septed hyphae was observed. A prolonged biotrophic phase was noted in cv Rosecoco-GLP-2 and line M29 and lesions appeared on the 6th and 10th days,  respectively, characterised by conspicously septed hyphae. Such septations were not observed in hyphae of the same isolate in slide culture. A transverse section of the infected tissue in cv Rosecoco-GLP-2 and line M29 taken 9 days after inoculation showed cell disintegration whereas the cells in line M26 were intact. The fungus only colonised the lower epidermal and spongy mesophyll layer. Sporuration in cv Rosecoco-GLP-2 and line M29 occurred 8 and 12 days after inoculation, respectively, but did not occur in line M26. The number of synnema per lesion and number of conidiophores per synnemata in cv Rosecoco-GLP-2 was significantly (P=0.05) higher than in line M29. Thus,  resistance in bean line M26 to angular leafspot is probably due to inhibited germination, colonisation and sporulation whereas the intermediate response in line M29 could be due to delayed and limited sporulation of the fungus.

Key Words:  Angular leaf spot, cellular reactions,  Phaseolus vulgaris, spore inhibition

RÉSUMÉ

Le développement fongique et des réactions cellulaires associées dans trois cultivars de haricot/lignée (Phaseolus vulgaris L.) inoculés avec l’isolat PG18 de Phaeoisariopsis griseola ont été étudiées.  Des differences dans l’extention du développement fongique et l’intégrité de la cellule-hote ont été utilisées pour comparer les réactions des trois cultivars/lignées (résistant, intermediare résistant et sensible).  Les conidies de P. griseola ont germé 4 heures après inoculation en produisant des tubes de germes soit à un, deux bourgeons ou bien aux deux cotés.  Cependant la germination dans la lignée M26 était partiellement inhibée et presque toutes les conidies n’ont pas germé 4 heures après inoculation.  La pénétration est apparue 24 heures après inoculation et était soit directe ou à travers les stomates. Des tâches brunes miniscules étaient observées sur la zone inoculée 4 jours après inoculation sur la lignée M26, alors que dans le cv Rosecoco-GLP-2 (sensible) et la lignée M29 (intermediaire résistante), des hyphes visiblement cloisonnées étaient observées. Une phase biotrophique prolongée a été observée chez le cv Rosecoco-GLP-2 et des lésions ont apparu entre le 6^eme et le 10^eme jour respectivement caractérisés par des hyphes visiblement cloisonnées.  Tels cloisonnements n’ont pas été observés dans les hyphes du même isolat dans la boite de culture. Une section transversale des tissus infectés du cv Rosecoco-GLP-2 et de la lignée M29, pris 9 jours après inoculation ont montré une désintégration  de la cellule alors que les cellules dans la lignée M26 étaient intactes.  Le champignon a colonisé seulement la partie épidermique inferieur et la couche de la mésophylle spongieuse.  La sporulation dans le cv Rosecoco-GLP-2 et la lignée M29 ont apparu 8 et 12 jours après inoculation alors qu’elle n’a pas apparue chez la lignée M26.  Le nombre de synnema par lésion et le nombre de conidiophores par synnemata chez le cv Rosecoco-GLP-2 étaient significativement (P<0.05) plus élevés plus que chez la lignée M29.  Ainsi, la résistance chez la lignée M26 à la cercosporiose angulaire des feuilles est problablement due à la germination inhibée, à la colonisation et à la sporulation alors que la réponse intermediaire dans la lignée M29 serait due à la sporulation limitée et retardée du champignon.

Mots Clés:  Cercosporiose angulaire des feuilles, réactions cellulaires, Phaseolus vulgaris, inhibition des spores

Introduction

Angular leafspot of beans (Phaseolus vulgaris L.) caused by Phaeoisariopsis griseola Sacc. is a disease of economic importance in Kenya, especially where ideal conditions for its  multiplication prevail (Mwang’ombe et al., 1994). This disease  has been reported in more than 60 countries worldwide and yield losses may reach 80% (Guzman et al., 1995). Resistance of beans to angular leafspot has been shown to be of the race-specific type (Mulindwa, 1980) and governed by either one, two, or  three independent factors (Barros et al., 1957; Cardona-Alvarez, 1958; Santos-Filho et al., 1976; Singh and Saini, 1980). This type of resistance is often complete (Eenink, 1976) but its  level may be influenced by genetic dosage effect (Dunn and Mamm, 1970), modifier genes (Rouselle, 1974) or the physiological age of the plant (Bartos et al., 1969). Extraneous factors such as soil temperature, air humidity or light intensities also appear to raise or lower the level of this resistance (Hubbelling, 1966; Walker and Williams, 1973). The presence of an intermediate resistance reaction rather than an immune reaction is probably due to the effect of any one or more of the above factors, which influence the level of monogenic resistance. Guzman et al. (1995) reported that host resistance to angular leafspot of beans appears to be partial rather than absolute but the actual genetic control, whether a multigenetic horizontal resistance or an oligogenic vertical resistance, remains to be verified by a genetic analysis in segregating progenies.  A coevolution of the two major groups of P. griseola isolates, Andean and Mesoamerican (with the Andean and Mesoamerican common bean gene pools, respectively) has the practical consequence that beans of a given gene pool are more resistant to P. griseola isolates isolated from beans of the other gene pool (Guzman et al., 1999). 

A late type of multicellular resistance as reported in the interaction between barley and Erysiphe graminis f.sp. hordei (Wei et al., 1994) may account for the intermediate resistance reaction in bean line M29 (Wagara, 1996). There are also indications that rate-reducing resistance against P. griseola occurs in some of the bean cultivars whereby cultivars show differences in the time when symptoms appear and the extent of disease severity (Buruchara, 1983). Reduction of the rate of infection reduces the epidemic development of the disease by decreasing the reproduction rate of the pathogen (Parlevliet, 1979). The factors of resistance which lead to the latter are; reduction in infection frequency or lesion numbers, lengthening of the latent period and the decrease in sporulation capacity. These aspects need to be considered and incorporated in future studies when attempting to develop stable resistance against P. griseola in beans. Thus, this study was carried out to establish the differences in pre-penetration and post-penetration events of P. griseola on bean cultivars/lines with different levels of resistance.

Materials and Methods

Isolation of the pathogen and inoculum preparation. Isolation was made from lesions on naturally infected bean leaves from Kabete Campus Field Station showing fungal sporulation  and was named isolate PG18. The fungus was cultured on Bean Leaf Dextrose Agar at 24ºC in darkness according to the method described by Karanja et al. (1994). A spore suspension was prepared from 14 day old cultures of P. griseola, conidial concentration determined using an improved Neubauer haemocytometer and adjusted to 2 x 10⁴ conidia ml^-1.

Test cultivars and plant inoculation. Three bean cultivars with different levels of resistance to  P. griseola were used.  Included were a resistant line (M26), an intermediate resistant line (M29) and a susceptible cultivar (Rosecoco-GLP-2). The seeds were potted in black polythene bags containing steam sterilised soil consisting of soil, sand, manure and ballast in the ratio of 2:1:1:1 in the glasshouse.

Inoculations were done on marked circles on both sides of the first and second trifoliolate leaves of 21 day old bean plants. Control plants were sprayed with sterile distilled water. Sections of leaf tissue were removed from beneath the inoculation droplets after 4, 8, 12, 48 hours and thereafter on a daily basis until the 15th day.

Light microscopy. Sections of leaf tissue removed from beneath inoculation droplets were cleared and stained using Bruzzese and Hasan clearing and staining technique (Bruzzese and Hasan, 1983). The sections were immersed in the clearing-staining solution consisting of 95% ethanol, chloroform, 90% lactic acid, phenol, chloral hydrate and Aniline blue in stoppered glass vials for 48 hours at room temperature. The tissues were then removed and placed for 48 hours in a concentrated chloral hydrate solution (2.5 g ml^-2 water) and rinsed in distilled water. They were mounted in clear lactophenol on a microscope slide for viewing under the light microscope. The number of synnemata per lesion and the number of conidiophores per synnema were determined. The values obtained were transformed using the square root transformation and thereafter analysed statistically.

Semi-thin sections (1.0-1.5 µm) of resin-embedded leaf tissue prepared as for transmission electron microscopy (O‘Connell et al., 1984) were  mounted and stained at 60 ¡C for one minute with 1% (w/v) toluidine blue in 1% aqueous borax solution. Viewing and photography were carried out under the light microscope.

Scanning electron microscopy (SEM). Infected bean leaf tissues were obtained as above and processed according to the method used by O‘Connell et al. (1984). The tissues were fixed in 2.5% (w/v) glutaraldehyde in 0.05 M sodium cacodylate buffer (pH 7.2) for one hour at room temperature and dehydrated in a graded ethanol series (50%, 70%, 80%, 96% and 100%). The tissues were then mounted on metal stubs and gold coated. The specimens were viewed under the SEM and photographed where appropriate.

Results

Pre-penetration and penetration events of the pathogen. The visual observations made under the light and scanning electron microscope showed that in both the susceptible cultivar (Rosecoco-GLP-2) and moderately resistant line (M29), spore germination occurred 4 hours after inoculation and the fungus produced germtubes at either one or both tips of the conidia while others emerged from the sides. In the resistant bean line (M26), most of the conidia had not germinated and the germinated conidia had shorter germtubes than those in either cv Rosecoco-GLP-2 or line M29. Penetration occurred 24 hours after inoculation in all the three cultivars/lines and was either direct or through the stomata. Some germtubes were observed to have penetrated directly just nearby a stoma.

Colonisation of the bean tissue. Observations made under the light microscope showed one or more primary hyphae growing out of infection vesicles and progressed both intercellularly and intracellularly. In cv Rosecoco-GLP-2 and line M29, very thick dark brown conspicuous septa were observed on both the primary and secondary hyphae four days after inoculation, while within the same period  minute brown flecks (small groups of dead cells) were visible on the chlorotic inoculated area of line M26. Tissue browning and granulation of the penetrated epidermal cells and a few of the adjacent uninfected cells was noted on the resistant bean line. Further observations of the inoculated tissues of cv Rosecoco-GLP-2 and line M29 under the light microscope showed a prolonged period of biotrophic development, and necrosis did not occur until the 6th and 8th day, respectively. In these two cultivars/lines, infected cells did not immediately become brown and lesions appeared after 6 days in cv Rosecoco-GLP-2 and 10 days in cv M29 associated with growth of a more extensive mycelia.

Six days after inoculation, development of more extensive hyphae with thick dark brown closely spaced septa spreading within and in between cells were quite conspicuous in cv Rosecoco-GLP-2 and line M29 as compared to line M26 where hyphal spread was limited but the septation was also conspicuous. This type of septation was not observed in Riddell slide culture of the same isolate where septa were far apart, thin and pale brown in colour. Observations made under the light and scanning electron microscope 9 days after inoculation showed that some of the conidia on line M26 had still not germinated. In cv Rosecoco-GLP-2, the inoculated area became pale brown in colour by the 5th day and observation under the light and scanning electron microscope revealed abundant growth of mycelia within the spreading lesion. The cells within the lesion were macerated and a transverse section taken on the 9th day showed cell disintegration.

Further observations made under the light microscope showed that colonisation of bean tissue by P. griseola was both intercellular and intracellular. Hyphae grew within host cells or along the middle lamella and progressed to the surrounding cells. Colonisation was confined more to the spongy mesophyll region whereas the cells towards the palisade region were intact although the upper leaf surface had already turned brown.

Sporulation. On the 8th day after inoculation, observations made under the light microscope showed that the brown, necrotic lesions on cv Rosecoco-GLP-2 had started sporulating, and dark clusters of conidiophores (synnemata) were visible on the under surface of the leaf. On line M29, smaller lesions than on cv Rosecoco-GLP-2 were visible on the 10th day but P. griseola did not sporulate until the 12th day. On line M26, the inoculated areas were  necrotic but no synnemata were visible 15 days after inoculation and the inoculated areas had restricted growth of conspicously septed hyphae. Observations under both light and scanning electron microscope on this bean line showed that some of the conidia of P. griseola had still not germinated on the 15th day after inoculation. Using the light and scanning electron microscope, the number of synnemata per lesion and number of conidiophores per synnema were established. The number of synnemata per lesion and conidiophores per synnema in cultivar Rosecoco-GLP-2 was significantly (P = 0.05) higher than in line M29 (Plate 1). The number of synnemata per lesion in cv Rosecoco-GLP-2 ranged from 30 - 52 as compared to 5 to 20 in line M29. Likewise, the number of conidiophores per synnema ranged from 48 to 106 in cv Rosecoco-GLP-2 and 15 to 43 in line M29 (Table 1).

Table 1.  Number of synnemata per lesion and number of conidiophores per synnema on cultivars Rosecoco-GLP-2, M29 and M26 fifteen days after inoculation with P. griseola isolate PG18

This study revealed that resistance to angular leafspot is not complete and involves either inhibition of conidia germination, colonisation,  sporulation or a combination of the three.  These results are in agreement with those obtained by Guzman et al. (1995) who reported that host resistance to angular leafspot appears to be partial rather than absolute. In line M26, resistance is due to an inhibition in conidia germination, limited colonisation and an inhibition in sporulation whereas the intermediate reaction in line M29 is due to delayed and reduced sporulation even though active lesions developed. Mwang’ombe and Shanker (1994) reported that the differences among cultivars indicating an initial stagnation of events and suppression of sporulation on resistant Coffee arabica genotypes could possibly be related to resistance to Colletotrichum coffeanum. The nature of the stimulus governing the direction of P. griseola germtube growth and penetration was not clear because some of the germtubes by-passed stomata and then penetrated directly. Dickinson (1949) suggested that the entrance through stomata may be a positive hydrotrophic response but not all plant pathogenic fungi   respond in this way. Many organisms that gain ingress by direct penetration of the cuticle of a leaf produce germtubes that grow directly over stomata but never enter the leaf through stomatal openings.

In all the 3 cultivars/lines, infection vesicles formed in living epidermal cells and primary and secondary hyphae developed to varying extents producing symptoms that ranged in severity from flecking to spreading lesions. The initial survival of the penetrated epidermal cells may reflect a ‘basic compatibility’ between P. griseola and its host species, perhaps due to a passive failure of the plant to recognise the pathogen or to the activity of other pathogen genes that regulate the invasion of living host cells. More commonly, fungal pathogens employ a combination of strategies, such as killing cells quickly to reduce the degree to which defences can be induced, and possessing attributes (such as enzymes that detoxify antimicrobial compounds) that mitigate the effects of those defence responses that take place (Heath, 1997). The subsequent biotrophic development of the pathogen terminated at various stages: it was short in line M26 and more prolonged in cv Rosecoco-GLP-2 and line M29. The duration of the biotrophic phase could be determined by specific recognition occurring at different times, thus, triggering premature host cell death and resistance. A prolonged period of biotrophic development is essential for the production of spreading lesions whereas when the biotrophic phase is of shorter duration or absent, premature death and browning of infected cells lead to restriction of both pathogen growth and extent of symptoms (O’Connell et al., 1985). Alternatively, in the absence of any specific recognition, the infected cells will survive initially and the duration of the biotrophic phase would then be determined by non-specific genes controlling various aspects of host and pathogen physiology. There is evidence that different resistance genes do operate at different stages of infection (Slesinski and Ellingboe, 1971) and that the expression of single resistance genes can vary according to the genetic background in which they occur. A quite different impendment to successful infection in the resistant cultivar may be the absence or scarcity in the host of nutrients essential for development of the pathogen. An impaired fungal nutrition as a resistance factor of induced resistance against biotrophic fungi has been described by Steiner and Sch’o’nbeck (1995).  There is increasing evidence that during the very first infection stages, Venturia inequalis relies on the degradation of the epidermal cell wall and later on active nutrient supply by the host (MacHardy, 1996). Keitt and Boone (1954) isolated mutants in the apple scab fungus Venturia inequalis whose requirements for vitamins, nitrogen bases, or amino acids were specific and pathogenicity was lost in some of the mutants unless the particular requirement was supplied with the inoculum.

According to the results obtained in this study, it is clear that bean line M26 is not extremely resistant to P. griseola because there was no rapid death and browning of penetrated epidermal cells and flecking only occurred 4 days after inoculation. Although much more limited, the infection vesicles were formed in line M26 just like in cultivar Rosecoco-GLP-2 and line M29. The most common response of resistant plants to cellular invasion by biotrophic or non-biotrophic fungal pathogens is rapid cell death (Heath, 1997).  In resistant cultivars of a host plant, this 'hypersensitive response' (HR) may be conditioned by  parasite - specific resistance genes but it also occurs following penetration of cells of non-host species in which such genes may be present (Heath, 1996). O’Connell et al.  (1985) reported that extreme resistance of bean cultivars to Colletotrichum lindemuthianum involved rapid death and browning of penetrated epidermal cells without formation of infection vesicles or a detectable biotrophic phase whereas extreme susceptibility involved the initial development of fungal infection vesicles within living epidermal cells followed by colonisation of further host cells by intracellular primary hyphae. From the foregoing account, it is apparent that generalisations on plant reactions based on macroscopic observations may be very misleading and cytochemical studies are necessary to determine the mechanism of resistance operating in each case.

The time and rate of sporulation of P. griseola was found to differ in the three bean cultivars/lines. In line M26, no sporulation was observed at all whereas limited and delayed sporulation occurred on line M29 as compared to cultivar Rosecoco-GLP-2. The reduced rate of epidemic development is in the case of partial or slow rusting resistance often explained by the combined effect of four components; infection frequency, latent period, infectious period and spore production (Neervoort  and Parlevliet, 1978). Studies on simulated epidemics suggest that from the components of partial resistance, latent period may be the more important one in determining the rate of epidemic build up with the more resistant cultivars tending to have a longer latent period than the susceptible ones (Zadoks, 1972). Apparently, the components of partial resistance may occur together to reinforce each other. For example in barley leaf rust, reduced sporulation rates per uredosorus are at least partially associated with longer latent periods because of the late start in sporulation (Parlevliet, 1986). This may be the case in line M29 where sporulation is delayed and limited. With pathogens increasing exponentially within the crop, the spores produced early after infection like in the case of cultivar Rosecoco-GLP-2 have a much greater impact on epidemic development than spores produced rather late after infection. In case of two cultivars having an identical total spore production per unit leaf area, the cultivar with the later onset of spore production (longer latent period) will be the more resistant one in the field.

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