Sclerotinia stem rot, a fungal disease caused by Sclerotinia trifoliorum, is often a serious problem in faba beans (Vicia faba). The levels of resistance to sclerotinia stem rot were evaluated in 23 faba bean cultivars originating from various European countries, 18 cultivars from ICARDA, five cultivars from Canada, 58 populations originating from various provinces of Greece, and five V. narbonensis populations. Evaluation was done under controlled conditions following artificial inoculation with carrot root pieces colonized by the fungus. Faba bean plants were scored for resistance on a 0 to 3 severity scale. There was significant variability for resistance to S. trifoliorum among faba bean cultivars and among populations. Seven cultivars and 15 Greek faba bean populations showed a satisfactory level of resistance (≤ 1.50), whereas 22 cultivars and 25 populations were susceptible (> 2.00). However, all V. narbonensis populations were resistant to S. trifoliorum (disease severity from 1.11 to 1.44) without significant variability within populations. Variability in the resistance of faba beans to S. trifoliorum would allow selection within appropriate genotypes for breeding in an effort to provide an effective alternative for sclerotinia stem rot management in this crop.
Évaluation de la résistance de la féverole à la pourriture à sclérotes causée par le Sclerotinia trifoliorum
La pourriture à sclérotes, une maladie fongique causée par le Sclerotinia trifoliorum est souvent un grave problème pour la production de féverole (Vicia faba). À la suite d’une inoculation artificielle avec des morceaux de carottes colonisés par le champignon, l’intensité de la résistance à la pourriture à sclérotes a été évaluée en conditions contrôlées chez 23 cultivars de féverole provenant de divers pays européens, 18 cultivars de l’ICARDA, cinq cultivars canadiens, 58 populations issues de diverses provinces de Grèce et cinq populations de V. narbonensis. Une cote de résistance a été attribuée à chaque plante sur une échelle d’intensité de maladie allant de 0 à 3. Il y avait des différences significatives pour la résistance au S. trifoliorum parmi les cultivars et parmi les populations de féverole. Sept cultivars et 15 populations grecques de féverole ont démontré un degré satisfaisant de résistance (≤ 1,50), alors que 22 cultivars et 25 populations étaient sensibles (> 2,00). Par contre, toutes les populations de V. narbonensis ont été résistantes au S. trifoliorum (intensité de maladie entre 1,11 et 1,44) sans qu’il n’y ait de différence significative entre les populations. Les différences de résistance au S. trifoliorum dans la féverole signifient qu’il serait possible de procéder à de la sélection au sein de génotypes choisis afin d’offrir l’amélioration génétique comme moyen efficace de lutte contre la pourriture à sclérotes de cette culture.
Corps de l’article
Sclerotinia stem rot, also known as white mold, is a serious disease caused by fungi of the genus Sclerotinia with a wide host range. It is considered a major disease of many important crops such as dry bean (Phaseolus vulgaris L.), soybean (Glycine max L.), alfalfa (Medicago sativa L.), and many other legumes (Delclos et al. 1997; Halimi and Rowe 1998; Kim et al. 2000; Steadman et al. 1997). This disease is often a serious problem of faba beans (Vicia faba L.) in Greece (Roupakias 1983) caused by the fungus Sclerotinia trifoliorum Eriks. (Lithourgidis et al. 2003).
Several studies, using different evaluation methods under controlled conditions, indicated measurable genetic differences in the levels of resistance to S. trifoliorum in alfalfa (Halimi and Rowe 1998; Pierson et al. 1997; Pratt and Rowe 1994; Rowe and Welty 1984) and red clover (Trifolium pratense L.) (Delclos et al. 1997). Similar data were reported for a closely related disease caused by Sclerotinia sclerotiorum (Lib.) de Bary in dry bean (Hunter et al. 1981; Miklas et al. 1992a, 1992b; Steadman et al. 1997) and soybean (Boland and Hall 1986; Chun et al. 1987; Cline and Jacobsen 1983; Kim et al. 2000; Nelson et al. 1991; Wegulo et al. 1998). Although partial resistance to sclerotinia stem rot has also been identified with field evaluations in several crops, the results of these evaluations are often inconsistent due to specific environmental conditions (low temperatures and high level of moisture) required for disease development and the high spatial variability associated with this disease (Delclos et al. 1997; Kim et al. 2000). Chun et al. (1987), and Nelson et al. (1991) observed significant differences among soybean cultivars for lesion length, but these results frequently were not correlated with field ratings. Boland and Hall (1986), and Cline and Jacobsen (1983) reported significant differences among soybean genotypes using the limited-term inoculation method under controlled environmental conditions. Wegulo et al. (1998) reported that, despite poor repeatability across experiments, the detached-leaf inoculation method in soybean had the greatest correlation with field ratings. Kim et al. (2000) reported a significant correlation between resistance detected with the infested-oat-seed inoculation method and field results in soybean. Pratt and Rowe (1994) demonstrated an increase in the resistance of alfalfa genotypes to sclerotinia stem rot in the field and the greenhouse following one cycle of selection using a stem-lesion bioassay.
Screening under controlled conditions may provide rapid and accurate evaluation of resistance of a large number of genotypes in a relatively small space with great reproducibility (Hunter et al. 1981; Lithourgidis et al. 1989; Pierson et al. 1997). Moreover, with measurements of field resistance, it is impossible to distinguish between the level of resistance and avoidance expressed by crop plants (Miklas et al. 1992b). Thus, screening under controlled conditions is often preferred by researchers for determining the level of resistance to sclerotinia stem rot in various crops.
Despite the great number of crops and methods tested for identifying resistance to sclerotinia stem rot, the level of resistance to S. trifoliorum in faba beans has not been investigated. Artificial inoculation with carrot root pieces colonized by the fungus has been found to be a suitable method for rapidly screening large plant populations for resistance to sclerotinia stem rot in faba beans (Lithourgidis et al. 1989, 1991). This method can provide a more accurate evaluation of the level of resistance in plants because it can distinguish plants that have disease escape mechanisms.
The objective of this research was to investigate variability among and within various faba bean cultivars and populations for resistance to S. trifoliorum following artificial inoculation with carrot root pieces colonized by the fungus.
Materials and Methods
Twenty-three faba bean cultivars originating from four European countries, 18 cultivars from ICARDA, Syria, five cultivars from Canada, 58 populations originating from 17 provinces in Greece, and five V. narbonensis (L.) populations (Tables 1, 2, and 3) were evaluated for resistance to S. trifoliorum after artificial inoculation under controlled conditions.
Pathogen, inoculation, and incubation
An infected faba bean plant was collected from field at the Fodder Crops Research Institute located in Larissa (39o38’ N, 22o25’ E), Greece and S. trifoliorum was isolated on potato dextrose agar (PDA). Carrot root pieces (3 x 4 x 5 mm) were autoclaved (at 120oC and 1.5 atm for 20 min) and placed on PDA cultures of the fungus prior to the sclerotia formation. After incubation at 20oC for 72 h, the colonized root pieces were used as inoculum (Lithourgidis et al. 1989, 1991). Faba bean plants of each cultivar or population were grown in pots under controlled conditions (19-21oC, 12 h photoperiod). Pots were arranged in a completely randomized design. At least 25 4-wk old plants of each cultivar or population were inoculated with the colonized carrot root pieces, on the stem at the second internode from the top. Immediately after inoculation, the stems were wrapped at the point of inoculation with a piece of wet absorbent cotton, and fixed in position with parafilm to protect the inoculum and keep it in contact with the stem. Subsequently, the inoculated plants were placed in a growth chamber at 19-21oC under fluorescent light with a 16 h photoperiod. The plants were sprayed with water three times daily to ensure adequate moisture. After 72 h of incubation, the inoculum was removed and the plants were examined for lesions. Plants with no lesions were inoculated again to ensure that there were no inoculation failures during the experimental procedure.
Each cultivar or population was evaluated based on the severity of the infection on each individual plant. A disease severity scale from 0 to 3 (Lithourgidis et al. 1989) was used as follows: 0 = no visible infection, 1 = early infection indicated by a small lesion (< 10 mm), 2 = moderate infection (10-20 mm), 3 = severe infection characterized by a large, water-soaked lesion (> 20 mm) or collapse of the stem. The severity scale was determined after long-term observations of the behavior of the disease in faba beans both in the laboratory and the field. Thus, plants with a disease severity rating of 1 grow normally (less than 5% mortality); plants with a disease severity rating of 2 may grow or die (about 50% mortality); and plants with a disease severity rating of 3 normally die (more than 95% mortality) (data not shown). Therefore, genotypes with a mean disease severity rating equal to or less than 1.50 were considered to be resistant; those with a rating between 1.51 to 2.00 were intermediate; and genotypes with a mean disease severity rating of more than 2.00 were considered susceptible.
Mean disease severity and standard error for each cultivar or population were calculated based on the disease severity in each individual plant which was considered as a replicate. Cultivars and populations were ranked according to the mean level of resistance as determined by the disease severity scale used.
Results and Discussion
There was significant variability in the level of resistance to S. trifoliorum among cultivars (Table 1) and Greek populations (Table 2) with disease severity of 1.18 to 2.73 in cultivars and 0.89 to 2.74 in Greek populations. Coefficients of variation in cultivars and populations were 0.20 and 0.23 respectively. Only seven of the cultivars and 15 of the Greek populations exhibited a high level of resistance to the disease (disease severity ≤ 1.50). Among cultivars, A-246, and among populations, E-101, showed the highest level of resistance with disease severity of 1.18 and 0.89 respectively. Regardless of the level of resistance, there was significant variability among cultivars and among Greek populations tested, as indicated by the high values of standard error (0.07-0.21). All of the V. narbonensis populations showed a higher level of resistance to S. trifoliorum, compared with cultivars and Greek populations of V. faba (Table 3). Disease severity in these populations was relatively low (1.11-1.44) and the coefficient of variation also was low (0.10).
The significant variability within faba bean cultivars and populations tested in this study would allow selection for resistant individuals within each cultivar or population, so it is not necessary to resort to V. narbonensis populations to find genes for resistance to S. trifoliorum. This is very important since all previous attempts to obtain interspecific hybrids between V. faba and V. narbonensis were unsuccessful (Lazaridou and Roupakias 1993).
Evaluation of dry bean cultivars under controlled environmental conditions indicated that there was also significant variability in resistance to S. sclerotiorum (Hunter et al. 1982). However, these cultivars were very susceptible to the disease. Only 6.5% of the cultivars were partially resistant. On the other hand, more than 15% of the faba bean cultivars tested in this study were resistant. The percentage of resistant Greek populations was much greater (25.8% of the populations tested). This may be attributed to the fact that the Greek populations, unlike the cultivars, have never been subjected to selection and, consequently, genes for resistance have not been lost. In the two neighboring provinces of Trikala and Karditsa in central Greece, none of the six populations evaluated were susceptible to the disease (Table 2). The widespread and continuous cultivation of other hosts, such as alfalfa and red clover, probably created an extensive infected area in which only the relatively resistant genotypes of faba beans eventually survived in local populations.
Miklas et al. (1992a), studying partial physiological resistance to white mold in dry beans, found significant differences in mean lesion length among small- and medium-seeded dry bean genotypes. However, in our study, seed size of faba beans was not correlated with the level of resistance to the disease (r = - 0.21, NS). Some populations with small seed size were susceptible (e.g., E-16, E-88, E-115), whereas others of the same group were resistant (e.g., E-27, E-105, E-107). Similarly, populations with large seed size were susceptible (e.g., E-92, E-109, E-110) or resistant (e.g., E-12, E-22, E-61).
The high variability observed within each cultivar, regardless of whether it was ranked as resistant, susceptible or intermediate, may be attributed to the fact that faba beans are about 35% cross-pollinated (Bond and Pope 1974). Thus, genotypes resistant to S. trifoliorum could be detected and selected at the individual plant level even within a susceptible cultivar. The differentiation among cultivars is maximum at 72 h (Lithourgidis et al. 1991), so any plant that shows resistance (disease severity 0 or 1) after more than 72 h of contact with the fungus should be considered resistant to the disease.
Field experiments to evaluate the productivity and the level of resistance of the selected cultivars did not show the same disease rating detected under controlled conditions, and this was probably the result of unfavorable conditions prevailing during these growing seasons (Lithourgidis 1991). Differences for resistance to sclerotinia stem rot disease between controlled and field conditions have been previously reported (Chun et al. 1987; Delclos et al. 1997; Kim et al. 2000; Nelson et al. 1991). Selection for resistance to sclerotinia stem rot in the field is difficult considering that the disease requires high moisture and moderate temperature, conditions that are not always present. Evaluation of the level of resistance in faba beans is therefore necessary under controlled conditions.
Data of this study provide an evaluation of the resistance variability of faba beans to S. trifoliorum under controlled conditions. To our knowledge, similar evaluation studies with faba beans do not exist in the literature. Considering the absence of research with faba beans in this area, these data might be useful for further research on resistance of this crop to sclerotinia stem rot. For this purpose, cultivars or populations with different levels of resistance could be crossed in order to study the inheritance of disease resistance genes, and to select high yielding and resistant genotypes.
The authors are grateful to Professor N. Fotiadis and Professor P. Gerakis, Aristotle University, Faculty of Agriculture, for their critical review of the manuscript.
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