Abstract: Isolates of Sclerotinia sclerotiorum were collected from infected lentil plants from 2 agro-ecological zones of Syria and used to study their comparative growth on culture media and pathogenicity on different lentil genotypes. The growth studies were carried out on Potato Dextrose Agar (PDA) growth media under laboratory conditions. Mycelial radial growth and sclerotial production were the parameters used to compare the isolates. Pathogenicity studies were carried out with selected isolates on 10 lentil genotypes, infected as detached shoots and as whole potted-plants in the plastic house. The isolates showed considerable variation in cultural characteristics through mycelial growth, mycelial pigmentation and sclerotial production in the media plates. There were significant differences in the growth and sclerotial production of most of the isolates, but no apparent correlation between mycelial growth and sclerotial production among the isolates. Genotype by isolate interactions was significant for the isolates tested for pathogenicity. These interactions, however, appeared to be caused by differences in virulence of the isolates and did not suggest the occurrence of distinct pathogenic races of the pathogen isolates.
INTRODUCTION
Lentil (Lens culinaris Medik.) is an important crop in the farming system of the Mediterranean region. About 650,000 tones of the pulse were produced in the Middle East and North Africa in 2001[1]. Its grain is used as human food to provide good-quality proteins to the resource-poor and its straw is an important feed source for small ruminants common in the region. Yields of the crop in farmers’ fields have remained fairly low at about 700 kg ha-1. Among other factors limiting yields are diseases. The most important disease of the crop in the region is vascular wilt caused by Fusarium oxysporum f.sp. lentis[2]. Screening techniques for this pathogen have been developed, resistant germplasm identified and control has been consistently obtained by the use of resistant cultivars[3]. The wilt/root rot complex is another important disease of lentil in the region and is becoming more severe on the crop. Control of this complex by the use of resistant cultivars however, has not yet been attained and other methods of management have to be considered[4-6].
Sclerotinia stem rot incited by Sclerotinia sclerotiorum (Lib.) de Bary is one of the important components of the root rot and wilt complex of lentil and other cool season legumes in the Mediterranean region[5]. Its incidence on the crop, especially when planted during winter months, is increasing due to heavy plant lodging from flush growth. The disease can affect the grain and biomass yields of the lentil, which is important for food and feed, respectively. Although yield losses from Sclerotinia stem rot have not been systematically estimated on lentil, incidences of up to 50% have been observed in some lentil trials and in farmers’ fields in the region[7].
S. sclerotiorum is primarily known as a pathogen of vegetables, oilseed and ornamental crops[8,9]. However, it also causes disease on forage legumes and cool-season food legumes such as chickpea, faba bean and lentil under favorable conditions[9]. On lentil, the disease is more severe on the winter-planted crop in the high rainfall agro-ecological zones of Syria. It has, however, also been observed on isolated fields in moderate rainfall zones even at higher incidence levels in some fields than the in the higher rainfall zones. This has led to the suspicion that there may be differences in the isolates of the pathogen occurring on the crop in the different zones.
On-going research at ICARDA to identify sources of resistance to S. sclerotiorum in lentil under controlled greenhouse conditions have only identified limited levels of resistance in a few lines evaluated[7]. Observed field reactions, however, on the natural incidence of the disease on lentil cultivars in farmers’ fields and on different lines in evaluation trials, tend to suggest that there may be some cultivar by isolate interactions. These observations also suggest that there may be some sources of multiple resistances to wilt and Sclerotinia stem rot pathogens in lentil, which may become available to growers in the near future. It is not known, however, whether host resistance developed in response to one isolate of S. sclerotiorum or in one location, will be effective against isolates or strains of the pathogen from other locations. No studies have been done to compare the growth of diverse isolates or their pathogenicity on lentil. The objective of this study was therefore to compare the growth and pathogenicity of isolates of S. sclerotiorum from different agro-ecological zones and locations in Syria, on selected lentil genotypes.
MATERIALS AND METHODS
Isolates: Ten isolates of S. sclerotiorum used in the growth studies were collected from diseased lentil plants during the cropping season of 1999, from different locations in 2 of the 3 agro-ecological zones of Syria, during routine disease monitoring of lentil on-farm trials. All the isolates originated from infected stem piece samples collected during the monitoring. Isolations from the infected samples were made by surface-sterilizing the pieces with 10% sodium hypochloride for 2 min, followed by three successive rinses in sterile distilled water. The samples were then blot-dried on sterile filter paper and plated on Potato Dextrose Agar (PDA) media and incubated on laboratory benches at 25-27°C. Isolates from the samples were purified by sub-culturing 3-4 days after plating and storing for later use.
Growth comparison: To compare the rate of growth among isolates, a 4 mm mycelial plug of a 2-day old culture of each isolate was placed in the middle of PDA media in a petri plate. Five plates were used for each isolate. The plates were incubated on laboratory benches and the mycelial radial growth rate of each isolate was measured on alternate days from day 3 following inoculation, up to day 7 after inoculation and just before the fast-growing isolates started to cover the plates. On the 9th day following inoculation of the plates when the fast-growing isolates had covered the plates and sclerotia formation started, a visual estimate of the indication of the sclerotial status of each isolate was recorded. Plates were incubated for a further 14-day period for sclerotial production, after which the number of sclerotia per plate and the average for each isolate was determined as another basis to differentiate between the isolates.
Pathogenicity of isolates on lentil genotypes: Four out of the ten S. sclerotiorum isolates evaluated for growth were chosen based on the diversity of place of collection and the observed differences in the growth and sclerotial production. These were used to make a comparison of their pathogenicity on lentil genotypes. Ten lentil genotypes were selected for use in the pathogenicity studies of the selected isolates. Four of the genotypes (ILL 5883, 7012, 7009 and 4400) were included because they were being evaluated in on-farm adaptation trials and had been observed to show varying reactions to S. sclerotiorum at some of the trials locations[7]. Two of the test entries (ILL 2130, 5588) were cultivars grown by farmers in the region and the other four (ILL 5722, 6819, 7005 and 6994) were promising lines under evaluation for different traits. Ten seeds of each of the genotypes were planted in each of 8 plastic pots filled with steam-pasteurized sand and soil mixture (1:1 v/v) and placed on plastic house benches. The plants were let to grow for 6 weeks after which two methods of inoculation (detached shoot and intact plant) were used to compare the reactions of the genotypes to the four selected geographical isolates of S. sclerotiorum based on lesion length development.
Detached shoot in Hoagland solution: Five stem shoots selected from one pot of each genotype on the basis of uniformity in length were detached for inoculation. Individual shoots (15-20 cm length) were supported by a cotton plug and the cut ends immersed individually in 1x14 cm test tubes containing Hoagland solution. Four of the five shoots were each inoculated by placing a 4 mm mycelial plug of S. sclerotiorum from a 2-day old culture grown on PDA, at the axil between the stem and the petiole at about the mid-portion of the shoot. The fifth shoot was inoculated with a plug of plain PDA and served as the control. Inoculated shoots were put on metal racks and placed in a growth chamber at 25-27°C and 80-90% relative humidity. Genotype shoots were completely randomized after inoculations before placement on the racks.
Intact plants in pots: Another set of 5 plants from the second pot of each genotype were selected based on the same criterion of stem length as before. Remaining plants in the pots were clipped and discarded. Four of the five plants selected, were inoculated by placing a 4 mm mycelial plug of S. sclerotiorum from the same source as with detached shoot inoculations, on the lowest axil towards the base of the plants in the pots. The fifth plant was inoculated with a plug of plain PDA and served as the control for each genotype. Each pot was covered with a transparent polyethylene bag for 24 h to increase relative humidity to above 80%. Pots were completely randomized on the plastic house benches after inoculations.
Disease assessment: Daily lesion development on the genotype shoots started 2 days after inoculations and was measured on each treatment as they progressed. This was continued until the first complete colonization of shoots when it was stopped for genotype rate comparisons. Mean daily lesion lengths on each genotype were calculated as the sum of the individual lesion lengths divided by the total number of inoculated shoots. At the end of the measurements, mean lesion lengths of each genotype were linearly regressed against time after inoculation, to determine the rate of lesion expansion, where the slope of the line represented the rate of lesion expansion (cm/day) on each genotype and inoculation treatment.
The pathogenicity test consisted of a 4x10 factorial experiment performed two times. The experiment consisted of the 10 lentil genotypes entries each inoculated with the four isolates of S. sclerotiorum. In each repetition of the experiment, four replicate stems of each treatment combination (entry x isolate) were arranged in a split plot design with genotypes as main plots and isolates as sub-plots. Analysis of Variance (ANOVA) was performed by use of Genstat statistical package. The similarities in entry responses to the isolates were evaluated by correlation analysis.
RESULTS
Morphology and cultural characteristics of isolates: All the ten isolates collected from diverse backgrounds and locations in Syria grew well on PDA media. There were significant differences (p<0.05) between some isolates collected from farmers' fields but not on those from research stations (Table 1). Isolates 20 and 30 collected from farmers’ fields, for example, had a lower mycelial growth rate than isolates 14 and 21 from other farmers’ fields. The number of sclerotia produced by each isolate after 24 days of incubation followed the same pattern. Isolate 30, which showed the least mycelial growth rate also produced the least number of sclerotia (Table 1). There was generally a poor correlation (r = 0.23) between mycelial growth rate and number of sclerotia produced by the isolates.
| Table 1: | Mycelial growth rate and sclerotial production of Sclerotinia sclerotiorum isolates collected from lentil in Syria |
| aSSL96- = Syria Sclerotinia on Lentil collected
in 1996 bF=Farmer’s field; R= Research station cMean from 5 culture media plates based on 7 days of growth dMean from sclerotial counts of 5 replicated plates |
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| Table 2: | Mycelial growth of ten Syrian isolates of Sclerotinia sclerotiorum from lentil on Potato Dextrose Agar |
| Means of five replications for each isolate. Means within each column followed by the same letter(s) are not significantly different at p=0.05 according to Duncan Multiple Range test | |
Significant differences were obtained in radial diameters of the test isolates. Mean radial diameter 7 days after incubation ranged from 2.6 to 7.0 cm. Isolate 14 produced the largest colony and isolate 30 had the smallest colony (Table 2).
Colonies of isolates on PDA ranged from white to a gray to a dark brown, with light tan being the most common. Colonies of isolates 17, 20 and 36 were wooly with well-defined zonation and those of 14 and 28 were tufted with faint zonation. The colony of isolate 21 had distinct concentric rings whereas that of isolate 30 was compact with no zonation. The bottom of colonies ranged in color from greenish gray to black. It was possible from these observations to relate cultural characteristics of the 10 isolates with specific groups based on virulence on lentil stems.
Isolate reactions on plants: Significant differences in virulence of isolates and in response of lentil entries were observed in both intact and detached shoot inoculations.
| Table 3: | Maximum lesion length (cm) on lentil stems inoculated with isolates of Sclerotinia sclerotiorum |
| Table 4: | Daily rate of lesion expansion on lentil stems inoculated with isolates of Sclerotinia sclerotiorum |
| *Determined by measuring the lesion expansion of each genotype over a 7 day period, Each value is the mean of 5 stem replications | |
There were significant differences in genotype reactions with each isolate.
For intact inoculations, isolate 36 was the most virulent, infecting all of the 10 lentil entries with an overall mean maximum lesion length of 5.0 cm as compared to an overall mean of 3.4 for isolate 17. Isolate 14 infected 9 of the 10 lentil entries, 17 infected 7 of the 10 and 20, 7 of the 10 (Table 3). Between lentil entries, ILL 7005 was the most resistant with a mean maximum lesion length of 0.9 cm among all the isolates while ILL 5722 was the most susceptible with a mean maximum of 7.3 cm. All other entries showed intermediate mean maximum lesion lengths ranging between the minimum and the maximum.
For detached shoot inoculations in trays, there was more virulence between isolates and among lentil entries than intact inoculations. All 4 isolates showed virulence to the 10 lentil entries with isolate 20 exhibiting the least mean maximum lesion length while the other 3 were similar. Within lentil entries, ILL 7012 was the most resistant with a mean maximum lesion length of 6.9 cm among all the isolates while ILL 7009 was the most susceptible with a mean maximum of 11.4 cm. The ranges were much closer with the other entries than observed with intact inoculations (Table 3). For each isolate, there were significant differences (p<0.05) between lentil entries in their mean lesion lengths, with isolate 17 showing the greatest virulence and 20 the least virulence (Table 3).
Rate of lesion expansion: There was less variation in mean rates of lesion expansion among isolates for inoculated and detached lentil shoots, as reflected by the isolate means (Table 4). Between lentil entries, there was more variation in isolate reactions in intact than in detached shoots.
Among the intact inoculations, the daily rate of lesion expansion ranged from 0.3 on ILL 7005 to 1.8 on ILL 5722. These were also the entries that produced the corresponding least and most maximum lesion expansion. There were significant differences (p<0.05) between entries inoculated with each isolate.
Among the detached shoot inoculations, the daily rate of lesion expansion ranged from 1.7 on ILL 7012 to 2.8 on ILL 7009, with less variation in isolate reactions between lentil entries. Isolate 17 showed the greatest virulence while 20 showed the least as reflected by their overall mean daily rates of lesion expansion among lentil entries.
DISCUSSION
The 10 Syrian isolates of S. sclerotiorum showed considerable variation in cultural characteristics through mycelial growth and sclerotial production. Four representative isolates also exhibited considerable variation in virulence and aggressiveness on 10 lentil genotypes when inoculated on intact potted plants and detached shoots in trays.
Isolate 36 produced the largest number of sclerotia in growth media under laboratory conditions. If sclerotial production is considered an important parameter in the virulence and perpetuation of Sclerotinia stem rot among lentil genotypes, then this isolate showed clear differences in its virulence among all the 10 isolates characterized.
ANOVA revealed no significant isolate by entry interactions, with the 10 isolates of S. sclerotiorum. This indicates that lentil entries were infected similarly by the four isolates of S. sclerotiorum selected and evaluated, despite the diversity in location of origin of the isolates.
The mean maximum lesion length results indicate that isolates 20 and 36 parasitized the lentil entries differently than did isolate 14 and 17 on the intact plant inoculations. Such differences could potentially indicate the occurrence of different pathogenic races, with 20 and 36 in one group and 14 and 17 in another. However, this did not appear to be the case when these same isolates were evaluated in detached shoots. Responses of lentil entries to isolates 14, 17 and 36 closely paralleled each other and only slightly different from the means of isolate 20, thus suggesting no distinct grouping into possible pathogenic races.
For all isolates, ILL 7009 was the most susceptible genotype, ILL 7012 was the least susceptible and most differences between the remaining entries were similar, when results of both intact and detached shoots were combined. However, the range of values for lentil entry means with isolate 36 for both maximum lesion length and daily rate of lesion expansion greatly exceeded the ranges for the other three isolates (Table 3 and 4). Therefore, the isolate by entry interactions for S. sclerotiorum appeared to be caused by differences in the ranges of values for lesion expansion among genotypes and not by contrasting interactions between isolates and genotypes that might suggest the occurrence of pathogenic races.
Results of this study did not demonstrate any clear differences between diverse isolates of S. sclerotiorum in virulence on lentil genotypes. Present data do not suggest that the 4 isolates can be assigned to distinct pathogenic race groups. Lentil entries differed in susceptibility to pathogen isolates and differences between the entries were generally consistent for the 2 methods of inoculation evaluated. Virulence of a pathogen may be determined by comparing the number of host genotypes on which isolates produce susceptible symptoms. In this study, isolate 36 was the most virulent; however, its severity of lesions varied among the genotypes with no consistent pattern in the 2 inoculation techniques to assign it to a distinct race group.
Further research would be useful to verify the principal results of this study. This could be with other isolates of S. sclerotiorum, collected from other locations and with other lentil genotypes among a population developed for resistance to the diseases. It could also be by the use of other techniques such as the detached shoot technique in Hoagland solution which has been established to be effective in differentiating chickpea reactions to S. sclerotiorum[10].