' F

4

n

PIant Patl?ology (2000) 49, 680-687

Characterization of pathotypes among isolates of Xanthomonas axonopodis pv. manìhotìs in Colombia j

I S. Restrepo, M. C. Duque and V.berdier* Biotechnology Resea;ch Unit, Centro Internacional de Agricultura Tropical (CIAT); and Institut de Recherch? pour le DBveloppement i

' I . (IRD), AA 6713, Cali, Colombia

Cassava bacterial blight, caused by Xanthonzoizas axonopodis pv. manihotis (Xam) is a destructive disease occurring in most cassava growing-areas. Although Colombian isolates of Xam differ in DNA polymorphism and pathogenicity, no suitable host differentials have been identified to demonstrate physiological specialization. A set of 26 Xaiiz isolates from three edaphoclimatic zones (ECZs) in Colombia was selected for inoculation on a set of 17 potential cassava differentials. Leaf inoculation and stem puncture were used in order to detect possible specific interactions between cultivars and isolates. Cultivar x isolate interaction was highly signijicant ( P < 0.001) after stem inoculation, but not after leaf inoculation. The stem inoculation technique was selected as a method for resistance screening of cassava cultivars for bacterial blight resistance. A highly significant interaction was also detected when cultivar behaviour was rated as area under the disease progress curve (AUDPC) after stem inoculation. Different pathotypes were defined among the 26 isolates and differential cultivars were proposed to define the pathotypic composition of Xam populations in three ECZs in Colombia. The results should help to improve selection of sources of resistance to cassava bacterial blight.

Keywords: cassava bacterial blight, pathotypes, virulence, Xanthonzonas axonopodis pv. iiianihotis

Introduction

Cassava (Manihot esculenta) is a major food crop in the tropics. In Colombia, efforts to develop high-yielding cultivars of cassava have been hindered by the suscepti- bility of several cultivars to cassava bacterial blight (CBB), a destructive disease in South America and Africa (Lozano, 1986).

The causal agent of CBB is Xanthomonas axonopodis pv. manihotis (formerly Xanthomonas campestris pv. manihotis; Vauterin et al., 1995), which can induce a wide variety of symptoms (Maraite, 1993). X . axono- podis pv. inanihotis ( X a m ) is a foliar and a vascular pathogen. No mechanism has been observed to limit the multiplication and development of the bacteria in the mesophyll of resistant cultivars during the foliar phase and intercellular multiplication in the mesophyll (Boher & Verdier, 1994), but defence mechanisms against the pathogen have been shown in the vascular system of infected cassava plants (Kpémoua et al., 1996).

~ The most appropriate and realistic approach for controlling CBB is through host resistance (Verdier et al.,

1 'To whom correspondence should be addressed. *E-mail: v.verdier@cgiar.org Accepted 27Jtlne 2000.

1997). Polygenic, additively inherited resistance has been developed from M. esculentu and the wild relative M. glaziovii (Hahn, 1978). Resistance can also be found in a genetically broad range of cassava accessions, and is not limited to one or a few 'lineages' of the host (Sánchez et al., 1999). Recently, a study under green- house conditions to understand the genetics of resist- ance to CBB in an Fi population of cassava allowed the characterization of 12 resistance quantitative trait loci (QTL) to five different Xam isolates (Jorge et al., 2000). Some of these QTL were associated with particular Xam isolates, suggesting a specific interaction between the plant and the pathogen (Jorge et al., 2000).

Effective breeding for resistance requires information on the diversity and geographical distribution of the pathogen. Considerable variation has been observed among Xam isolates in relation to biochemical and physiological characters (Fessahaie, 1997; Grousson et al., 1990), serology (Wydra et al., 1998) and genomic characters, as analysed through restriction fragment length polymorphism (RFLP) (Restrepo & Verdier, 1997; Verdier et al., 1993, 1998) or amplified fragment length polymorphism (AFLP) techniques (Restrepo et al., 1999a). Recent studies detected a high level of DNA polymorphism in isolates from South America (Restrepo & Verdier, 1997; Verdier et al., 1998;

680 02000 BSPP

Pathogenic variation of the CBB pathogen 681

Restrepo et al., 1999b). In Colombia, Xam isolates collected from three edaphoclimatic zones (ECZs) are geographically differentiated, and no migration of isolates occurs between ECZs (Restrepo & Verdier, 1997). However, within an ECZ the same haplotypes were found at different sites, showing that at this level migration of isolates occurs at a high rate (Restrepo & Verdier, 1997).

Differences in virulence among X a m isolates were first described by Robbs et al. (1972). Virulence variation was also observed among isolates from Brazil (Takatsu et al., 1978; Alves & Takatsu, 1984) and Africa (Maraite & Meyer, 1975; Grousson et al., 1990; Fessehaie, 1997). Fifty-two isolates collected in Colom- bia from different locations were grouped into four groups of virulence after inoculation onto three culti- vars (Anonymous, 1987). Isolates also showed differ- ences in the speed of symptom development, suggesting variation in aggressiveness (Verdier et al., 1993, 1994). More recently, 10 pathotypes were defined among 91 Xam ísolates in Venezuela, using five cassava cultivars as differentials (Verdier et al., 1998). Mutations to change virulence are considered to occur readily among Xanthomonas spp. (Stolp et al., 1965), and probably explain the occurrence of CBB epidemics in several cassava-growing regions (Wydra et al., 1998). Other results suggested that an isolate x cultivar interaction also exists (Maraite & Meyer, 1975; Maraite et al., 1981). However, a larger number of cultivars and a wider range of isolates should be evaluated to confirm these results (Maraite et al., 1981), as a clear-cut classification by race is not observed because of the lack of host differentials and insufficient knowledge of genetics of resistance to CBB.

The work reported here was conducted to investigate further the nature and extent of pathogenic variation in populations of X a m in Colombia, using a larger number of cultivars and a wide range of isolates. The present study took advantage of previous work on the molecular characterization of the host (Sánchez et al., 1999) and on the genetic composition of the Colombian Xam populations (Restrepo & Verdier, 1997; Restrepo et al., 1999a). Its objective was to characterize the specificity of various resistance sources to Colombian isolates of Xam from different ECZs. To this end, specific goals were: (i) to define inoculation methods suitable for assessing virulence and aggressiveness; (i;) to investigate the presence of cultivar x isolate inter- actions in infection experiments of 17 cassava genotypes with 26 Xum isolates; and (iii) to derive from this data potential differential host sets and pathotypes.

Materials and methods

Cassava cuitivars

Based on a previous study (Sánchez et aZ.; 1999), 17 cultivars from the cassava germplasm collection at CIAT were selected as potential host differentials for

CBB. They were chosen for their different geographical - origins and adaptability to the biotic and abiotic conisolatets in the main edaphoclimatic zones (ECZs) where cassava is cultivated. They were also chosen for theif distribution in the different ECZs favouring the more widely used. The two parents (MNGA-2 and CM2177-2) of the cross used by Jorge et al. (2000) that permitted the characterization of specific QTL were included. Cassava accessions were coded as either CG (cassava genetics programme, controlled cross) or CM (cassava improvement programme, controUed cross). Landraces were coded by country of origin (MVEN, Venezuela; MCOL, Colombia; MBRA, Brazil; MNGA, Nigeria). All cassava plants used for the inoculation experiments were grown from mature stem cuttings in sterile insectisoil collected in Santander de Quilichao (Colombia), as previously described (Verdier et al., 1998). This soil is characterized by a texture with up to 10% organic matter, low pH (4-5-5-0), low contents of P, Ca and Mg, and the presence of free Mn and Al. 'c

Bacterial isolates

Twenty-six isolates, representing the 26 RFLP haplo- types described in Colombia in 1996 (Restrepo & Verdier, 1997), were used for inoculation. These 26 haplotypes belonged to different genetic groups having distinct RFLP patterns, using the pthB gefe as a probe (Verdier et al., 1998). For long-term storage, isolates were kept in'60% glycerol at -80°C. Isolates were plated on YPG medium (5 g L-' yeast extract, 5 g L-' peptone, 5 g L" glucose and 15 g L-' agar), and grown for 24 h at 28°C before inoculation.

Inoculation techniques

Inoculations were conducted in a greenhouse at 28I19"C (dayhight temperatures), under a 12-h photoperiod and 80% relative humidity. Four cultivars were selected to compare leaf and stem inoculations. They included the two parents, CM2177-2 and MNGA2, of the cross used by Jorge et al. (2000), and two cultivars widely used in ECZ 1 (MCOL22) and ECZ 2 (CMS23-7). Cultivars CM523-7 and MCOL22 permitted the identification of pathotypes in Venezuela in a previous study (Verdier et al., 1998). The stem inoculation experiment was extended to the remaining 13 cultivars. All plants were 1-month-old at the time of inoculation.

Leaf inocdation The third and fourth leaves from the apex of three plants per genotype were inoculated by placing 10 gL of a bacterial suspension (10' cfu mL-') in a small hole (2-mm diameter) previously punched out with a cork borer. The small diameter of the hole permitted the inoculum to stay on the lesion until the total infiltration of the bacteria into the leaf. Control leaves were treated with sterile water. Angular leaf spots around the hole were observed 7 days after inoculation. For each .

O 2000 BSPP Plant Pathology (2000) 49, 680-687

I *’ h

682 S. Restrepo et al.

cultivar x isolate combination, the average size of the six lesions was estimated by scanning the lesions with a video system (Stratagene-Stratagene Inc., Eagle Eye II still video system, La Jolla, CA, USA) and calculating the area with the SCI-SCAN software (Anonymous, 1993; Kirchhof & Pendar, 1993).

Stem inoculation Çassava stems were inoculated by inserting a toothpick, contaminated by passing through a 24-h-old culture of the appropriate isolate, between the third and fourth leaves from the top. This protocol resulted in about lo7 CFU mL-’ of stem extract (G. Sanchez, S. Restrepo and V. Verdier, unpublished data). Five replicate plants of each genotype were inoculated with each of the 26 isolates. Inoculated plants were arranged in the greenhouse according to a randomized complete block design with five replicates. Non-inoculated plants were included in each experiment as controls. The entire experiment was replicated twice.

Symptoms were monitored 7, 14 and 30 days after inoculation, and disease was rated according to the following scale: O = healthy plant, no reaction observed; 1 = dark area or necrosis around the inoculation point; 2 = gum exudates on the stem; 3 = wilting of one or two leaves and exudates; 4 = more than two leaves wilted; and 5 = complete wilting and dieback. Disease reactions O to 3 were considered incompatible (resist- ance), while reactions 4 and 5 were considered com- patible (susceptibility). Virulence or avirulence was rated based on the proportion of virulent reactions over all replicate plants. An isolate was considered virulent on,a cultivar if it generated symptoms in classes higher thän 3 in at least one plant. Furthermore, the area under disease progress curve (AUDPC) was calcu- lated for each inoculated plant from the disease reaction scores 7, 14 and 30 days after inoculation for each cultivar-isolate combination as:

AUDPC = &.[(Di + Di- 1) * ( t i - ti- 1)]/2,

where D is the disease score using the 0-5 severity scale, and t corresponds to days after inoculation, with i = 7, 14 or 30 days (Shaner & Finney, 1977).

AUDPC were used because they allow comparison of the aggressiveness of isolates to the various cassava genotypes, and the relative resistance of each plant genotype to the different isolates. In this study, ‘virulence’ refers to the ability of an isolate to infect a host andiusually, to reproduce on it (Andrivon, 1993). ‘Pathotype’ is a group of isolates sharing a common phenotype of virulence to a set of host cultivars (Caten, 1987). ‘Aggressiveness’ refers to the severity of disease induced by an isolate for a particular host-pathogen interaction (Andrivon, 1993).

Statis tical analyses

Stem inoculation experiments The disease reaction scores after 30 days were analysed

with the Kruskal-Wallis nonparametric variance analysis PROC NPARlWAY (SAS Institute Inc., Cary, NC, USA) (SAS, 1989aJb). AUDPCs were calculated for each plant before .being averaged over all replicates within a cultivar .X isolate pair. The resulting AUDPCs were log- transformed, and subjected to analysis of variance (ANOVA) with isolates, cultivars and the ECZs of origin of the isolates as fixed factors. Duncan’s multiple range test was used to compare mean values of the variable log (AUDPC) (P < 0.05) for each facto5 isolates, cultivars, ECZs and the interaction isolate x cultivar.

Leaf bzoculation experiment The areas of leaf lesions were analysed by ANOVA with isolates and cultivars as fixed factors. Duncan’s multiple range test was used to compare mean values of the variable areas of leaf lesions (P < 0.0.5) for each factor and .the interaction isolate x cultivar. Spearman’s rank correlation was used to compare leaf inoculations with log (AUDPC) data from stem inoculations. ANOVA were performed using the GLM procedure of SAS with. plants as replicates.

Results

Virulence of IL2m isolates and pathotype determination

Stenz inoculation The data from both experiments were analysed together, as the ANOVA showed no significant effect of replications in time on disease scores (F = 0-9, P = 0-03). Interactions between 26 Xanz isolates and 17 cassava genotypes were highly significant (Kruskal- Wallis test; P < 0.001). Nineteen different pathotypes were identified among the 26 isolates (Table 1). None of the pathotypes constituted more than 15% of the sample and most of them were represented by only one isolate (Table 1).

According to results of the stem inoculation experi- ments, MBRA886, MBRA685, MBRA902, MNGA2 and CM6438-14 were the most resistant cultivars (resistant to more isolates), but resistance was incom- plete. CM2177-2, CG402-11, MVEN2.5, MCOL1.522 and MCOL2261 were clearly the most susceptible cultivars (susceptible to all isolates), and the remaining cultivars showed a broad range of reactions (Table 1). In terms of virulence, measured as the number of cultivars showing a resistant reaction to a particular isolate, the reaction of the different isolates also varied greatly. ECZ 1 isolates were the least virulent, inducing fewer susceptible reactions on all cultivars. The average number of virulent reactions induced by ECZ 1,2 and 5 isolates was 12-6, 14 and 14-8, respectively.

Interactions between isolates and cultivars adapted to the ECZ of origin of the isolates were studied. Five different pathotypes were defined among the eight ECZ 1 isolates, according to the reactions of six culti- vars adapted to this ECZ (Table 2). The five cultivars

O 2000 BSPP Plant Pathology (2000) 49, 680-687

I o !2

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Table 1 Pathotypes of Xanthomonas axonopodis pv. manihotis detected by stem inoculation among 26 isolates collected in three edaphoclimatic zones (ECZs) in Colombia. Numbers below cultivar . indicate their ECZ.of adaptation

.- ~

ö i 3

E o_ CM2177-2 CM523-7 MNGA2 MCOLSb MCOL1522 MVEN25 MERA881 MCOL2261 MERA886 MERA685' CM5286-3 MCOL1505 CG402-11 MERA902 CM6438-14 MERA12 MNGA79

. : Cultivar -. W

3-

Pathotype Isolateb ECZC Za 2 1 1 5 1 6 5 6 1 2 1 5 2 2 1 4 - E 1 CIO-167 2 Sd S S S S S S s . s S S s . S S S S S

CIO-174 2 a (D CIO-12 5

O v

$ 2 O

IYJ m -l

CIO-22 5 s CIO-46 2 CIO-1 2

CIO-37 2

CIO-119 2 s CIO-136 2

CIO-121 2 s CIO-24 1 S CIO-40 2 S CIO-62 1 S

CIO-4 2 S CIO-25 1 S

CIO-33 2 S CIO-151 2 S

CIO-171 2 S CIO-64 1 S CIO-59 1 S CIO-5 2 S CIO-81 1 s CIO-11 5 CIO-168 2 S CIO-84 1 S CIO-90 1 s

S S S S S S S S S S S S S R S S

3 S S S S S S S S. R S S S S S S S

S S S S S S R S S S S S R S S R S S R S S S S S R S S S S S S S S R S S R S S

S S S S S S S S s S S S S S S S S S S S S S S S S S

S S S S S S S S S S S S S

S S S R R S

R S

R R R S S

S S S S S R S R

R R R S R

S S S R S S S R S S S S S

S S S S S S S S

S S S S S

S S S S s S

S S S S S S S

R R S R s R S S R S R S S S S R S S R S

R R R

S R

s ,

S S S S S S S S S S S S R S S S S S S S S S R S S R

4 5

6 7

8 9 10 11 12 13

14 15 16

S R S S S S S S S S S S S

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S S R

R . S S R S S R R S

S S S S S R

S S S

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R S R

S S R

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17 18

19

'Restrepo & Verdier, 1997. bCIO, CIAT-ORSTOM Xanthomonas axonopodis pv. maniholis collection at Biotechnology Research Unit, CIAT, Cali, Colombia. 'ECZ1, subhumld tropics: ECZ 2, acid soil savannas: ECZ 5. high-altitude tropics. dA cultivar was consldered susceptible if it generated symptoms in classes 4-5 in at least one out of 10 plants.

684 S. Restrepo et al.

A Table 2 Pathotypes defined after the analysis' of the reactions from stem inoculations between isolates and cultivars originating from the same edaphoclimatic zone (ECZ). The haplotype, determined by RFLP, of each isolate is indicated in parentheses

Cassava cultivars

ECZ Pathotypes (haplotypesb) MERA685 MVEN25 MBRA12 MNGA2 MCOL22 MCOL1505 Isolates

1 c1-1 '210-24 (CIO). 62 (C15) Sa S S S S S c1-2 CIO-25 (Cll), 81 (C5) R S S R S S CI-3 CIO-59 (C14). 64 (C16) R S S S S S CI-4 CIO-84 (Cl8) S S S R S R CI-5 CIO-90 (C6) R S R R R R

CM2177-2 CM523-7 CM5286-3 MERA902 CM6438-14

2 c2-1 CIO-1 (Cl) 37 (C2). 40 (C12), 46 ('213) S S S S R C2-2 CIO-4 (C3) S S S R S c2-3 CIO-151 (C20) S S R S R C2-4 CIO-5 (C4), 121 (C21) . S S S R R C2-5 CIO-119 (C19), 33 (C22). 136 (C17), S S S S S

167 (C23), 168 (C24), 171 (C25), 174 (C26)

CG 402-11 MCOL1522 MCOL2261

5 C5-1 CIO-Il (C7), 12 (Ca), 22 (C9) S S S

RFLP, restriction fragment length polymorphism. aA cultivar was considered susceptible if it generated symptoms in classes 4-5 in at least one out of 10 plants. bHaplotypes are designated according to Restrepo & Verdier (1997), based on RFLP patterns of total genomic DNA generated with the pthB probe.

Table 3 Mean areas of leaf lesions (in mm2) induced on six leaves of four cassava cultivars after inoculation with Xanrhomonas monopod~s pv. manihotis isolates

ECZa Isolateb

Cultivars

CM523-7 MCOL22 MNGA2 CM2177-2 . Mean'

I .

i. '

2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 5 5 5 1 1 1 1 1 1 1 . 1 Mean' f SE

CIO-I CIO-4 CIO-5 CIO-33 CI037 CIO-40 CI046 CIO-119 CIO-121 CIO-136 CIO-151 CIO-167 CIO-168 CIO-I71 CIO-174 CIO-11 CIO-12 CIO-22 (210-24 CIO-25 CIO-59 CIO-62 CIO-64 CIO-81 CIO-84 CIO-90 45.4 1: 3.2 a

. 28.3 175 37.7 34.1 37-7 34.7 460 350 50.7 34.6 49.4 43.0 39.6 40.0 45.5 44.7 38.8 33.8 42.6 35-7 568 ' 390

' p9.9 ' . 41.1 ' ,34.8 31.2

50.3 39.4 . ,37.2 32.0

35.5 27.3 44.3 . 36.6

. .55.2 32.7 :'51.4 406 "'.. .37.4 44.2

' , 58.7 33.0 :489 367

. I " ,529 46.9 : :51.9 49.1 : "464 31.7

52.6 33.4

~. .. . , ". , .

2:

-- 36.4 i 2.7 b 38.3 f 4.6 b

22 1 40.7 406 38.7 48.8 31-5 54.5 469 34-2 336 59.0 28.8 32.5 35.0 54.4 25.9 31.4 50.9 33.6 37.6 31.0 41.7 61.3 20.8 41.3 199 180 i 1.6 c

11.7 17.8 168 16.8 11.9 15.9 16.4 159 15.9 17.1 18.4 21.7 179 19.3 188 16.8 20.2 19.0 16.9 19.7 22.3 23.2 12.8 15.4 20.3 31.4

, i

20.6 d 326 abc 33-1 abc 34.1 abc 36.5 abc 35.2 abc 357 abc 37.5 abc

32-1 abc 41.9 a 34.6 abc 35.6 abc

39.5 abc

30.4 bcd

30.1 bcd

29.3 cd 28.3 cd 37.7 abc 34.6 abc 37.3 abc 33.7 abc 39-2 abc 38.0 abc 35.7 abc 40.6 ab 33.5 abc

'ECZ, edaphoclimatic zones; ECZ 1, subhumid tropics; ECZ 2, acid soil savannas; ECZ 5. highaltitude tropics. bCIO, ClAT - ORSTOM Xanthomonas collection, Biotechnology Research Unit, Cali, Colombia. 'Means followed by the same letter are not significantly different (P= < 0.05).

O 2000 BSPP Plant Pathology (2000) 49. 680-687

Pathogenic variation of the CBB pathogen 685

adapted to ECZ 2 allowed the identification of five pathotypes among the 15 isolates from this ECZ, while all three isolates from ECZ 5 belonged to the same pathotype according to their reaction on the .three cultivars adapted to this ECZ (Table 2).

CÒmparison of the isolates for aggressiveness

Leaf symptoms The average size of leaf lesions varied widely among and within, cultivars (Table 3). ANOVA revealed significant effects of cultivar' (F = 88.62; P < and isolates ( F = 2.22; P = but no significant cultivar x isolate interaction ( F = 1.17; P = 0-1944). The mean squares for cultivars were higher than those for isolates, showing that there was more vari:ation between the four cassava cultivars than between Xum isolates. The com- parison of means by cultivars and by isolates is shown in Table 3.

Disease progress afier stem inoculation Isolates, cultivars and the isolate x cultivar interactions had an effect on aggressiveness measured as the disease progress after stem inoculation. ANOVA of the AUDPC indicated significant differences between cultivars and isolates, and a significant interaction between these two factors (P < 0.001) (Table 4). Although cultivar-iso- late interactions were significant for disease severity, their contribution was much lower than that of cultivars and isolates separately (Table 4). The mean squares for cultivars was higher than that for isolates, showing that there was more variation between cassava cultivars than between Xam isolates. The ANOVA also showed signifi- cant differences when isolates of the three ECZs were separated ( P < 0.01) (Table 4). Duncan's multiple range test showed that no significant difference was observed between isolates from ECZs 2 and 5, whereas isolates collected in ECZ 1 differed significantly in their reac- tion response from isolates collected in other ECZs.

Isolates appeared ro- differ in aggressiveness, with CIO 12, CIO 167, CIO 22, CIO 40 and CIO 46 being the most aggressive (data not shown). Isolate CIO-90

Table 4 Analysis of variance of area under disease progress curves (AUDPC) using general linear model. Components of variance are the cultivars, isolates, and the edaphoclimatic zone (ECZ) in which isolates were collected

. AUDPC~ . . .

Source . d.f. MS Fvalue P-value

Cultivar f6 11.6 27-38 0.0001 Isolate .' 25 499 11.79 0~0001 Cultivar x Isolate 400 0611 1.45 10~0001 ECZb 2 086 4.97 00074

d.f. = degrees of freedom; MS = meani square Transformed to log (AUDPC). bECZ where isolates were collected.

was the least aggressive. However, the mean comparison test did not identify any particular group as being signi- ficantly different, continuous variation being observed (data not shown).

The AUDPC was highest when MCOL2261 was inoculated with CIO 168, CIO 136, CIO 12 and CIO 22, confirming the susceptibility of this cultivar. Between the highest value [log (AUDPC) = 4.5151, obtained for MCOL2261 and isolate CIO 168, and the lowest one [log (AUDPC) = 1-93], obtained for CM6438-14 and isolate CIO 24, a continuum of values was observed, showing reaction- diversity between ïsolates and cassava cultivars (data not shown).

Comparison of aggressiveness to leaves and stems

Isolates were ranked according to aggressiveness mea- sured as either leaf lesion areas or AUDPC following stem inoculation on four cassava cultivars. Spearman's rank correlation coefficient test showed no significant correlation (2 = 0.07; P = 0.75) between these two criteria for assessing aggressiveness.

Discussion The present study analysed host-pathogen interactions within a wide range of cassava cultivars and of Colom- bian Xam isolates. Two inoculation methods were assessed. Although we did not compare the leaf and stem reactions for all 17 Cultivars, the information we obtained on the subset of four cultivars showed that leaf reactions were not correlated with resistance as mea- sured by stem reaction. Stem inoculation seems suitable to use to screen for resistance rapidly and efficiently and to detect isolate x cultivar interactions. The lack of correlation between leaf and stem reactions is probably due to the fact that resistance mechanisms occm in the stem vascular tissues. Indeed, parenchyma cells in the phloem or adjacent to the xylem play an important role in resistance, synthesizing callose and phenolic com- pounds such as lignin (Kpémoua et al., 1996). More- over, bacterial lysis pockets in the xylem limit or stop bacterial blight extension (Kpémoua et al., 1996).

The use of one isolate per haplotype group for detecting isolate x cultivar interactions has been recom- mended (Restrepo & Verdier, 1997) and has proved useful in characterizing X a m pathotypes (Verdier et al., 1998; and this study). Recently in Colombia, the detection of new haplotypes permitted the detection of new pathotypes but also new haplotypes that also appeared recently presented a pathotype previously described (S. Restrepo and V. Verdier, unpublished). One hypothesis to explain this finding in the Xaml cassava pathosystem is that the genetic variation detected with RFLPlpthB is evolving more rapidly than the virulence phenotypes.

A high level of pathogenic diversity exists within each ECZ, confirming recent results that showed a micro- geographic distribution of genetic diversity in Xam

,

8 2000 BSPP Plant Pathology (2000) 49, 680-687

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686 S. R e s t r q o et al.

(S. Restrepo, C. M. Velez and V. Verdier, unpublished data). Furthermore, the set of bacterial isolates used in this study is sufficiently pathogenic to overcome the resistance of a large number of the cassava cultivars used currently. This result highlights the need to search for new sources of resistance. This study showed that a wide range of host resistance and pathogenic variation exists in all ECZs, and that this variation is not correlated to the ECZ. Therefore, resistance sources can be selected in all cassava germplasm and not only in the set of genotypes adapted to some ecological conditions. For example, cultivar MBRA886, adapted to ECZ 6, showed a high level of resistance to isolates collected in ECZ 1 and 2. The results also provide a basis on which to recommend some cultivars for developing new crosses and thus generating progenies with broader disease resistance. Cultivars MBRA685 and MNGA2 showed a high level of resistance to most of the ECZ 1 pathotypes. Cultivars CM6438-14 and MBRA902 showed resistance to some pathotypes defined in ECZ 2. However; cultivars resistant to the pathotype from ECZ 5 have not yet been characterized. The high level of aggressiveness showed by the ECZ 5 isolates suggests a need for characterizing more suitable sources of resistance.

The definition of pathotypes and the selection of differential sets in each ECZ are based on the overall cassava breeding strategy developed in CIAT, which is based on improving varieties for each of the ECZs (Anonymous, 1983). The use of a differential host system specific to each ECZ and the identification of resistant cultivars to an ECZ population of the patho- gen will allow breeding for ecological adaptation and resistance at the same time. In addition, successful breeding of cultivars durably resistant to Xam will depend on adequately characterizing virulence in pátho- gen populations in target environments, the ECZs. As pointed out by Mew (1987), applying a single differ- ential system to tally with the pathotypes of pathogens present in highly diverse agro-ecosystems is very diffi- cult. The incorporation of cultivars widely used in the same ECZ of different countries would allow the com- parison of the pathotypic composition of Xam popula- tions at the level of the ECZ. Cultivars MCOL22, MCOL1505 and CM2177-2 proved to be useful host differentials, and permitted the identification of patho- types in ECZs 1 and 2 in Colombia (this study) and in ECZ 1-2 in Venezuela (Verdier et al., 1998).

Pathotype groups were defined and differences in aggressiveness were shown using 17 cassava cultivars. This may help to identify isolates useful for screening cassava lines for resistance. To screen promising cassava cultivars for release in a specific ECZ, isolates that represent the different pathotypes are recommended.

The AUDPC permitted the aggressiveness of isolates to . be determined. The quantification of the stem inoculation results using the AUDPC allowed variance analysis to be completed. The disease index as a categorical variable was not suitable for variance

analysis. However, the AUDPC data not only showed the existence of specific isolate x cultivar interactions but also established the geographical differentiation of the isolates' aggressiveness.

Based on the study of the pathotypic diversity of Xam in Colombia, an overall strategy for the identification and further characterization of pathotypes and resistant cultivars in different ECZs at a country level is proposed. Because of the long crop cycle of cassava and the problems in the availability of cassava plants, it is difficult to inoculate a large collection of isolates. Therefore, it is proposed to use RFLP-based markers to select isolates among bacterial populations showing high levels of genetic diversity and then to screen cultivars and select them according to their differential reactions to the isolates. Finally, a set of isolates representing the pathotypes characterized at an ECZ level are recommended for screening resistance to CBB.

Acknowledgements

We thank B. Boher, C. Bragard, E Correa and E. Duvellier for critically reading the manuscript and for their helpful comments. We thank M. Levy for his helpful suggestions in analysing the data and interpret- ing results. We gratefully acknowledge W. Roca (CIAT), J. Tohme and E. Alvarez for their support. We thank A. González, J. Valencia, and B. Pérez for their'.help in greenhouse experiments. We also thank E. de Páez for editing. This research was supported by grants from IRD and CIAT and by a doctoral fellowship awarded to S. Restrepo by IRD.

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