Evaluation of a collection of faba bean (Vicia faba L.) genotypes
originating from the Maghreb for resistance to chocolate spot (Botrytis fabae) by assessment in the field and laboratory
A. Bouhassan
1,∗, M. Sadiki
2& B. Tivoli
31Laboratoire de Biologie et Physiologie V´eg´etale, Facult´e des Sciences Ain Chock, Universit´e Hassan II, BP 5366, Maârif Casablanca, Maroc;2D´epartement d’Agronomie et d’Am´elioration des plantes, Institut Agronomique et V´et´erinaire Hassan II, B.P. 6202, Rabat, Maroc; 3Institut National de la Recherche Agronomique de Rennes, Station de Pathologie V´eg´etale, Domaine de la motte, BP 29, 35663, Le Rheu Cedex, France; (∗author for correspondence; e-mail: bouhassan@hotmail.com)
Received 7 January 2003; accepted 23 September 2003
Key words: Botrytis fabae, chocolate spot, resistance, screening,Vicia faba
Summary
Screening of 136 faba bean lines for resistance to chocolate spot caused by the fungusBotrytis fabaewas conducted in field conditions with artificial inoculation. Detached leaves of these genotypes were also assessed for disease severity after inoculation under controlled conditions with aB. fabaesuspension. The BPL710 and Aguadulce genotypes were inserted as resistant and susceptible checks, respectively. Disease symptoms were scored visually in both experiments. Diameter of lesions and production of spores were measured in the detached leaf assay.
The disease score index and diameter of lesions were used to calculate the area under the disease progress curve (AUDPC) to measure the disease progress. Significant differences were detected among genotypes for reaction to the disease in the field. However, no complete resistance was observed. For highly susceptible and highly resistant genotypes, the laboratory assay generally led to the same result as the field test, nevertheless, for more intermediate levels the agreement was weak. On detached leaves, diameter of lesions was significantly correlated to the AUDPC of disease scores (r = 0.89) and moderately correlated to sporulation (r = 0.52). The correlation between field AUDPC and detached leaf AUDPC of disease scores was low (r = 0.26). A low correlation was also observed between field AUDPC and lesion size (r = 0.30) and between field AUDPC and sporulation (r = 0.32). Among the 136 genotypes evaluated in this study, nine were convincingly and highly resistant in both tests. FRYM167 and FRYA58 genotypes were the most resistant with low AUDPC of disease scores, low AUDPC of lesion diameter and low spore production. These genotypes obviously have partial resistance.
Abbreviations:AUDPC – Area Under the Disease Progress Curve; ICARDA – International Center for Agricultural Research in the Dry Areas
Introduction
Chocolate spot, due to the fungusBotrytis fabae,is the most destructive leaf disease of faba bean crops in the world. This pathogen can reduce yields by more than two thirds (Sundheim, 1973; Hanounik, 1981). In the Maghreb region the losses can reach 80% of the annual production under optimum conditions for the develop-
ment of the pathogen. In Morocco, the yield reduction is assessed up to more than 60%, and occasionally the disease can induce a total crop failure under heavy epidemic infestation (Mabsoute & Saadaoui, 1996).
Although chemical control may provide partial protection, it is costly for small farmers, reduces the crops profitability, and is harmful to the envir- onment. Therefore, selecting resistant varieties is an
1994).
In spite of the efforts devoted to faba bean ger- mplasm collection and evaluation in the Maghreb, very limited work has been devoted to screening this material for disease resistance (Sadiki & Halila, 1998).
Hence, the objective of this research was to identify and evaluate sources of partial resistance to this patho- gen in a broad-based collection of faba bean acces- sions adapted to the Maghreb environment. Screening was conducted in the field and on a detached leaf as- say. This last test was also conducted to investigate two components of resistance to chocolate spot disease and to determine the correlation between detached leaf assay and whole plant reactions.
Materials and methods
The evaluation of a faba bean collection was con- ducted in the 1998–99 season. It consisted of two experiments carried out in the field and in the laborat- ory. The field experiment was conducted with artificial inoculation of the whole plant, while the laboratory evaluation consisted of inoculating detached leaves from the same faba bean lines.
Plant material. The plant material included 136 faba bean lines that originated from the Maghreb region.
These lines were drawn as representative core acces- sions from the faba bean collections held in Morocco, Tunisia and Algeria (Sadiki et al., 2002). The line, BPL 710, rated as resistant by ICARDA (Hanounik
& Maliha, 1986) and cv. Aguadulce were used as resistant and susceptible checks, respectively.
covered with 5 to 10 ml of sterile water. The spores were dislodged from the surface of the agar, by passing gently an elbowed Pasteur pipette (Tivoli et al., 1986).
The substance obtained was filtered through two layers of sterile gauze and diluted with tap water. The spore concentration was adjusted by using a hemacytometer slide.
Field screening. The experiment was laid out on 8 January 1999 as a randomised complete block design consisting of two replicates at SOGETA (Société de Gestion des Terres Agricoles) experimental station, Ain Dick, Rabat. Ten seeds of each faba bean line were planted in single rows 1.00 m long and 0.50 m apart. The checks were inserted every sixth line. Plants were inoculated two months after sowing (8 March 1999) by spraying the foliage with 15 to 20 ml of the inoculum per plant. The concentration of spore sus- pension was 3×105spores/ml. To enhance the disease development, a 2nd inoculation was applied 15 days later. High humidity was maintained by sprinkling the plants with a fine vapour of water for 60 minutes, two times a day, until the symptoms appeared extensively on the susceptible lines. The experiment was irrigated as needed.
Disease symptoms were scored weekly on the basis of a 5-class visual scale (Tivoli, unpublished), with: 1 = no symptoms or very small spots; 2 = very small and discrete lesions; 3 = some coalesced lesions with some defoliation; 4 = large coalesced sporulat- ing lesions, 50% defoliation, some plants dead; 5 = extensive lesions on leaves, stems and pods, severe defoliation, heavy sporulation, blackening and death of more than 80% of the plants. Scoring commenced a week after the first inoculation and was repeated for
Figure 1. Disease progress curves for two resistant, checks and two susceptible faba bean genotypes compared to the mean of 136 lines in a field assay under inoculation withB. fabae.
six dates until 5 May 1999. Two notes were taken, on an individual plant basis and on the whole row basis.
Laboratory experiment. In the laboratory assay, the plant material was prepared by growing the 136 lines and the checks BPL710 and Aguadulce, in an exper- imental field at the Hassan II Institute of Agronomy and Veterinary Medicine, Rabat. The lines were ran- domly planted as in the previous field test, but were not inoculated. Leaflets were detached from six-week- old healthy plants. Five leaflets were detached from the 4th node from the apex on the main stem of five random plants per line. Leaflets of two lines were dam- aged during their preparation for inoculation, thus the number of lines was reduced to 136 including checks.
Leaflets were immediately placed on a moistened fil- ter paper laid on sterile benches. The cut end of each leaflet petiole was covered with moistened cotton to maintain leaves at maximum turgor.
Inoculation was applied by dropping 20 µl of a conidia suspension containing 3×106 spores/ml on each leaflet. The benches were then covered with transparent polyethylene film held above the leaves and supported by frames to conserve maximum hu- midity. The incubation temperature was maintained to 20±2◦C.
The disease evolution was recorded every 24 hr by scoring the disease symptoms using a 9-class scale (Gondran, 1977) for nine days. Scoring commenced 24 hr after inoculation.
The diameter of emerging lesions was measured during this period. Number of spores was determined 11 days after inoculation on three random leaflets per line. These leaflets were each put in a fixed volume of sterile water and the number of spores per leaflet was estimated using a Malassez cell.
Statistical analysis. The disease symptom scores and measures of lesion diameter were used to calculate the area under the disease progress curve = AUDPC, ac- cording to the following formula (Shaner & Finney, 1977):
AUDPC=n
i=1
1/2
(yi+1+yi)(xi+1−xi) yi= disease severity at the day i (visual score or lesion diameter)
xi= time (days)
n = total number of symptom observations
The analysis of variance (ANOVA) for both field test and laboratory experiment was conducted with geno- type as the fixed factor for AUDPC values and number of spores (Dagnelie, 1984). Genotype means were classified and compared to the check means using LSD test.
Results
Field screening. Chocolate spot lesions appeared three days following the first inoculation. The overall
obtained the 3rdtime after inoculation (NL3), for 136 genotypes and the two checks inoculated withB. fabae.Dotted hyperboles indicate confidence belts at 95%. The individual genotype means are marked as circles.
score of a given bean line and the mean of its indi- vidual plants were highly correlated (r = 0.89, p = 0.001) in this material.
The progress of the symptoms evolved slowly on certain genotypes but at a much greater rate on other genotypes (Figure 1). The disease progress profiles of the resistant and susceptible checks were similar up to the 3rd time of scoring. From this time the sever- ity of the disease did not increase significantly on BPL710 but continued to increase on Aguadulce. The genotypes FRYT44 and FRYT19 suffered the most rapid development of symptoms (highest AUDPC) ex- pressing the highest score at the 3rd time of scoring.
Conversely, FRYM167 developed the least symptoms.
The maximum variation between resistant and sus- ceptible genotypes (score 1 and 5) appeared at the 3rd scoring date. The correlation between the mean disease scores at this date and the AUDPC values for all genotypes was high and significant (r = 0.91,p <
0.01) (Figure 2).
Disease scores at the third scoring date and the AUDPC values varied considerably among genotypes and statistically significant genotypic differences were observed (Table 1). The resistant check BPL710 was rated 1.9 with an AUDPC of 112 while Aguadulce, the susceptible check, was rated 2.9 and expressed an AUDPC of 177. The majority of AUDPC values were between 160 and 220 (Figure 3). Twenty-three geno- types were rated between 1 and 3 at the 3rdtime as well as at the 6th scoring time, with AUDPC values less
Figure 3. Frequency distribution of the AUDPC values for the data from the field experiment of 136 faba bean genotypes and the two checks inoculated withB. fabae.
than 160. Among these, eight genotypes had AUDPC values lower than the value expressed by BPL 710.
Fifteen genotypes expressed AUDPC values between 160 and 177 with the same average disease scoring (2) at the 3rdtime, but around or above score 4 at the 6th time of scoring. The remaining 98 entries had higher AUDPC values than Aguadulce.
Detached leaf test. Small lesions characteristic of chocolate spot appeared at the inoculum application site six to eight hours after inoculation. As they en- larged with time these small spots fused to form larger lesions, the severity of which varied according to lines.
The mean of the scores indicated that the discrimina- tion among the lines was significant three days after inoculation based on the lesion visual score and five days after inoculation based on the lesion diameter.
Figures 4 and 5 show the progression with time of disease score index and size of lesions for resistant and susceptible checks, two extreme lines and the over- all experiment mean. Five days after inoculation, the genotypes FRYM167 and FRYA27 showed the low- est disease score index (3.8), the same value as that recorded on the resistant check three days after inocu- lation. The genotypes FRYT44 and FRYT55 recorded
Figure 4. Disease progress curves for the two checks and the extreme lines (FRYT44 and FRYM167) compared to the mean of 136 genotypes in a detached leaf assay under inoculation withB. fabae.
Figure 5. Lesion diameter progress curves for the two checks and the extreme lines (FRYT44 and FRYM167) compared to the mean of 136 genotypes in a detached leaf assay under inoculation withB. fabae.
a disease score index of 7.4 four days after inoculation, whereas the susceptible check recorded a similar score six days after inoculation.
The genotype FRYM167 showed the smallest le- sions (7.7 mm), significantly smaller than those ex- pressed by the resistant check BPL710 (13 mm).
Highly significant differences were detected between genotypes for AUDPC scores, AUDPC diameter of lesions as well as for spore production (Table 2).
AUDPC of visual scores was highly correlated to the AUDPC of diameter of spots (r = 0.89,p =0.001).
Sporulation was significantly associated with the two
other parameters, the two correlation coefficients were identical (r = 0.52,p=0.001).
The AUDPC values of the disease scores and le- sion sizes of twenty-five genotypes were smaller than those for the susceptible check (54.0 and 172.5, re- spectively). Four lines showed very low AUDPC val- ues (29.8 to 31.7) and very low sporulation (1.6 to 2.7×105spores / leaflet). Altogether, seven genotypes were more resistant than the check BPL710.
The range for spore number per leaflet was from 0.9×105 (FRYT26) and 1.1×105 (FRYA7) to 25.7×105 (FRYT35). FRYM167 showed a similar sporulation as the resistant check (2.5×105 and
Genotype Disease score Lesion diameter Sporulation Mean AUDPC Mean AUDPC (105)
FRYA27 3.8 29.8 10.2 79.6 1.6
FRYM167 3.8 30.9 7.7 60.5 2.5
FRYA60 3.9 31.2 12.9 103 1.8
FRYA36 3.9 31.7 9.2 72.8 2.6
FRYA28 4.0 32.5 9.6 77.2 1.3
FRYT57 4.3 34.9 12.4 99.1 12.2
FRYT20 4.4 35.3 12.8 98.3 13.2
BPL710 4.4 36.0 13.0 104.4 2.1
FRYA10 4.5 36.0 10.6 84.2 1.2
FRYA29 4.5 36.7 11.2 86.9 7.8
FRYT13 4.6 36.5 10.3 81.3 3.2
FRYA32 4.6 37.5 11.2 88.3 2.9
FRYT58 4.6 37.6 14.3 113.6 5.2
FRYA7 4.7 38.3 12.5 99.0 1.1
FRYA51 4.8 38.5 12.2 97.4 4.6
FRYA62 4.8 38.8 12.4 96.4 7.7
FRYA42 4.8 39.4 14.0 112.7 2.6
FRYA41 4.8 38.7 13.8 108.2 8.1
FRYA56 4.9 40.5 11.0 88.1 1.5
FRYT24 5.0 39.8 15.2 121.3 2.5
FRYA25 5.0 40.2 13.8 108.1 14.7
FRYA59 5.0 40.5 12.8 98.9 13.5
FRYT26 5.0 40.7 14.2 115.4 0.9
FRYA39 5.0 41.2 13.5 108.5 6.6
FRYT14 5.1 41.5 16.0 128.0 3.5
FRYA58 5.1 42.0 12.0 96.0 4.7
Aguadulce 6.7 54.0 21.3 172.5 12.7
Meana 6.0 47.7 18.3 146.4 9.7
Min 3.8 29.8 7.7 60.5 0.9
Max 7.4 61.3 30.8 251.8 25.7
LSD (0.05) – 2.9 – 11.5 5.3
Meana= average of 136 entries; Max = the maximum value of 136 entries; Min = the minimum value of 136 entries. LSD = Least Significant Difference (p=0.05).
Comparison of the two tests. The analysis showed a significant correlation between the AUDPC of disease score index of the two tests (r = 0.26, p = 0.002) (Figure 6). Similarly, the correlations between field AUDPC and the two components AUDPC of diameter of lesions and sporulation were statistically signific- ant (r = 0.30, p <0.001 and r = 0.32,p =0.0001, respectively).
Discussion
The inoculation in the field screening was success- ful as all plants were infected with occasional hot – spots simulating the action of a natural outbreak. Fur- thermore, the susceptible and resistant checks were significantly different for their reaction to the disease but did not express the extreme reactions. Indeed, some lines were more susceptible than Aguadulce and several were more resistant than BPL710. The latter line was described as having a durable resistance based on multilocation testing in Asia, Europe and North America (Jellis et al., 1982; Hanounik, 1983; Bond et al., 1994).
The genotypes varied in reaction to the disease, but while some lines showed high level of resistance, none was completely resistant. This result suggests the pres- ence of a quantitative resistance in these genotypes (Parlevliet, 1979). The Botrytis fabae (BFIAV99-1) isolate used for the inoculum showed a significant virulence and pathogenic effect without destructive aggressiveness. Indeed, a very aggressive isolate does not allow the expression of small differences of reac- tions between genotypes (Parlevliet, 1983). Applica- tion of a single virulent isolate inoculum, instead of
Figure 6.Association between the AUDPC values obtained in the field screening experiment (AUDPCC) and the AUDPC values obtained in the detached leaf test (AUDPCNF) for 136 genotypes inoculated withB. fabae.Dotted hyperbole’s indicate confidence belts at 95%., observed means of the AUDPC for the resistant genotypes FRYM167, FRYA58 and FRYT14, for the two checks and for the most susceptible genotype FRYT44.
a mixture of isolates with a wide range of variation for virulence, can avoid confusion between vertical resistance and horizontal resistance (Parlevliet, 1983;
Hanounik & Maliha, 1986). To ensure the reliability of the field screening, only one isolate was used.
The visual score index at the 3rd week after in- oculation in the field was sufficient to rank the gen- otypes for their reaction to the disease. This result is in agreement with the study reported by Hanounik
& Robertson (1988). Additionally, the AUDPC val- ues clearly confirmed the ranking of the lines based on the score at week three. The AUDPC parameter presents the advantage of including the disease evol- ution. The use of this criterion rather than a single score at a specific stage may prove to be of import- ance under fluctuating climatic conditions influencing the development of the disease. In addition, the stage three weeks after inoculation may coincide with a dif- ferent seasonal period under different environmental conditions depending on planting date.
The laboratory test generally resulted in the same ranking as the field test for highly susceptible and highly resistant genotypes, but for more intermediate levels there was no clear agreement. Similar results were reported by Tivoli et al. (1986). This method partly suppresses the intrinsic physiological reaction of the plant and its reaction with the environment by removing the leaves. Nevertheless, this test has been used successfully to assess reactions of faba bean gen-
otypes toBotrytis fabaeby several authors (Shaaban
& Harrison, 1981; Hanounik & Maliha, 1986; Ha- nounik & Robertson, 1988; Tivoli et al., 1988). Thus, the detached leaf test can be very useful for under- taking the first screening of a large number of lines and thereby eliminating highly susceptible genotypes before conducting costly field tests. The difference in reaction of faba bean genotypes to chocolate spot by the two screening tests is undoubtedly due to the fact that each method permits expression of some particu- lar components of resistance more clearly than others (Tivoli et al., 1986). Genotype reactions under field conditions were not strongly correlated to the average lesion size and to the number of spores in detached leaf assay. However, the combination of field disease score and lesion diameter resulted in better discrimin- ation between lines. Thus, the laboratory assay seems to have potential for use in studies of the components of partial resistance.
The heterogeneity revealed, based on the spore number, indicates that the type of resistance of some lines acts on slowing down the pathogen phases without affecting the sporulation. This component of partial resistance (Johnson & Taylor, 1976; Parlevliet, 1979) does not appear to be an adequate criterion for interpreting the responses of faba bean lines to chocolate spot. Additional experiments are necessary to confirm such a result.
Acknowledgements
This paper is part of the result of a collaborative pro- ject FRYMED with inputs of the IAV Hassan II, Rabat, Morocco. The authors wish to acknowledge support received from M. Loutfi for his help in performing the experiments and N. Bourhim for the reading the concepts of this article critically. The authors like to thank M. Abou El Ouafa and H. Bouhya for their help in the field.
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