Characterization of Colletotrichum lindemuthianum races infecting dry edible bean in North Dakota

Abstract

Anthracnose, caused by the fungal pathogen Colletotrichum lindemuthianum (Sacc. & Magnus) Lams.-Scrib., is an economically important and damaging disease of dry bean (Phaseolus vulgaris L.) that can cause large reductions in yield and seed quality. Yield losses can reach 100% when contaminated seed is used, large amounts of inoculum are present, and favourable weather conditions occur during the crop cycle. Although widespread losses to anthracnose have not been observed in North Dakota, the lack of host resistance and favourable environmental conditions could lead to substantial economic losses. Additionally, numerous races of this pathogen exist and the pathogen race structure has the ability to change over time. Of the several races detected in North Dakota from 2003 to 2009, race 73 was the most prevalent. The purpose of this study was to determine the pathogen race types of C. lindemuthianum collected from dry bean samples in North Dakota from 2012 to 2014. Based on the 33 isolates collected in 2012 and 53 isolates collected in 2014, race 73 continues to be the most common race of C. lindemuthianum in North Dakota. Races 9 and 72 were also identified; however, these pose little additional threat due to virulence pattern similarities with race 73.

Résumé

L’anthracnose, causée par l’agent pathogène fongique Colletotrichum lindemuthianum (Sacc. & Magnus) Lams.-Scrib., est une maladie importante sur le plan économique ainsi que dévastatrice qui s’attaque aux haricots secs (Phaseolus vulgaris L.) et qui peut engendrer de lourdes pertes de rendement et altérer la qualité des semences. Les pertes de rendement peuvent atteindre 100% lorsqu’on utilise des semences contaminées, qu’elles comportent de grandes quantités d’inoculum et que des conditions météorologiques lui sont favorables durant le cycle de culture. Bien que des pertes généralisées causées par l’anthracnose n’aient jamais été observées au Dakota du Nord, le défaut de résistance de l’hôte et des conditions environnementales propices pourraient engendrer des pertes économiques substantielles. En outre, il existe de nombreuses races de cet agent pathogène et sa structure de race peut se transformer au fil du temps. De toutes les races détectées au Dakota du Nord de 2003 à 2009, la race 73 était la plus courante. L’objectif de cette étude était de déterminer les types de races de l’agent pathogène C. lindemuthianum collectés sur des échantillons de haricots secs au Dakota du Nord en 2012 et 2014. En se basant sur les 33 isolats collectés en 2012 et sur les 53 collectés en 2014, la race 73 demeure la race de C. lindemuthianum la plus courante au Dakota du Nord. De plus, nous avons identifié les races 9 et 72; toutefois, ces dernières ne constituent pas une menace supplémentaire compte tenu des similitudes qu’elles partagent avec la race 73 quant au patron de virulence.

Keywords:

• anthracnose
• Colletotrichum lindemuthianum
• dry bean
• Phaseolus vulgaris
• race identification

Mots clés:

• Anthracnose
• Colletotrichum lindemuthianum
• haricots secs
• identification de la race
• Phaseolus vulgaris

Introduction

Anthracnose, caused by the fungal pathogen Colletotrichum lindemuthianum (Sacc. & Magnus) Lams.-Scrib., is a damaging disease of dry bean that can cause blemishes on the seed, large reductions in yield, and seriously affect the market value of the seed (Mohammed & Sangchote 2007; Markell et al. 2012). This disease can affect all foliar parts of dry bean plants. Symptoms include long, angular, and brick-red to purple lesions that follow the leaf vein. Lesions appear on the abaxial side of the leaf more often than the adaxial side as a result of wind-dispersed or rain-splashed conidia coming in contact with the underside of the leaf first (Schwartz et al. 2005). Older lesions turn dark brown to black and appear sunken. The stems and petioles also can be affected (Allen & Lenné 1998). Pod lesions appear circular, sunken, and dark coloured surrounded by a red halo and defined raised edge with many acervuli containing masses of conidia (Tu 1988).

Anthracnose occurs worldwide, but causes greater yield losses in the temperate and subtropical climates as compared with tropical climates (Balardin et al. 1997). Relatively cool and humid conditions favour the development of this disease. Yield losses can reach 100% when contaminated seeds are planted, a large amount of inoculum is present, and favourable weather conditions occur during the crop cycle (Holliday 1980). Dry bean anthracnose is primarily managed through the use of clean seed along with crop rotation, application of fungicides, and the use of resistant cultivars.

Dry bean anthracnose was first detected in North Dakota in 1982 from seed submitted to the North Dakota State University Plant Disease Diagnostic Laboratory (Venette & Donald 1983). In 2011, 20% of dry bean fields in Wells, Eddy, and Ramsey counties had some level of anthracnose (Markell et al. 2012). While widespread losses due to the disease have not been observed in North Dakota, the lack of host resistance to common races and favourable environmental conditions could still potentially lead to substantial economic losses.

Pathogen races are determined by screening isolates against a standard set of 12 race differentials with diverse origins, each containing one or more of the major resistance genes (Table 1). A binary system is used to classify races of C. lindemuthianum (Melotto et al. 2000). Succinctly, each differential is assigned a discrete numerical value; the sum of the differentials with a susceptible reaction results in the C. lindemuthianum race designation (Pastor-Corrales 1991). For example, susceptible reactions on differentials ‘Michelite’ (value = 1), ‘Cornell 49-242’ (value = 8), and ‘Mexico 222’ (value = 64) would assign the isolate to race 73.

Table 1. The 12 differential lines used for race identification with the corresponding binary numbers, resistance genes and reactions to Colletotrichum lindemuthianum races 9, 72 and 73.

Differential cultivar	Binary no.^a	Resistance gene^b	Gene pool^c	Races
				9	72	73
Michelite	1	Co-11	MA	S	R	S
MDRK	2	Co-1	A	R	R	R
Perry Marrow	4	Co-1^3	A	R	R	R
Cornell 49-242	8	Co-2	MA	S	S	S
Widusa	16	Co-1^5	A	R	R	R
Kaboon	32	Co-1^2	A	R	R	R
Mexico 222	64	Co-3	MA	R	S	S
PI 207262	128	Co-3^3, Co-4^3	MA	R	R	R
TO	256	Co-4	MA	R	R	R
TU	512	Co-5	MA	R	R	R
AB 136	1024	Co-5, Co-6, co-8	MA	R	R	R
G 2333	2048	Co-3^5, Co-4^2, Co-5^2,	MA	R	R	R

^aBinary number is equal to 2ⁿ, where n is equal to the place of the cultivar within the series (Melotto et al. 2000).

^bResistance genes are according to the Bean Improvement Cooperative (http://bic.css.msu.edu/_pdf/Bean_Genes_List_2014.pdf).

^cGene pool: MA represents Mesoamerican, A represents Andean.

Using this nomenclature system, many races of anthracnose have been identified worldwide. Mexico reported the existence of 38 races, seven races were identified in Nicaragua, 33 races were identified in Colombia, and at least six have been reported in the USA (Balardin et al. 1997; del Río et al. 2002; Goswami et al. 2011). These reported races can be divided into two major groups: those occurring over wide geographic areas and those restricted to a single country (Melotto et al. 2000). Those present over a wide geographic area include C. lindemuthianum races 7, 65, 73. It is unknown whether this is due to repeated evolution of the pathogen race or to efficient seed-borne dispersal (Pastor-Corrales & Tu 1989). Research indicates C. lindemuthianum races are more diverse in Central America than either South or North America, which is consistent with other research reporting that pathogen races were more diverse in Mesoamerica compared with those from the Andean region (Pastor-Corrales 1996; Balardin et al. 1997; Sicard et al. 1997). This may be due to the origin of the wild bean ancestor in the Andean region and Central America and the coevolution of the pathogen and the host.

In North Dakota, C. lindemuthianum races 7, 73 and 89 were detected in 1994 (Kelly et al. 1994; del Río et al. 2002). New races detected in a North Dakota dry bean survey conducted from 2003 to 2009 included 1153 and 1161 – this was also the first report of these races in North America (Goswami et al. 2011). Among these, race 73 was the most commonly detected (Goswami et al. 2011). The appearance of new races may result in a change in race prevalence across the region. In Manitoba, Canada, race 105 has been detected and has a virulence pattern similar to race 73 except that race 105 is also virulent on ‘Kaboon’ which contains Co-1² (Dongfang et al. 2008). Currently, there are no races in North Dakota that overcome Co-1²; however, the previously detected races have the capability to infect dry bean cultivars grown in the area with varying degrees of disease severity (Goswami et al. 2011). Therefore, it is important to assess the races present throughout the state.

The objective of this study was to determine the pathogen races of C. lindemuthianum among isolates collected in North Dakota from 2012 to 2014. This evaluation will determine if the race structure of the C. lindemuthianum population in North Dakota has changed since the last evaluations were performed on isolates collected from 2003 to 2009 (Goswami et al. 2011).

Materials and methods

Field sampling

In the summers of 2012, 2013 and 2014, a foliar disease survey was conducted, and samples of infected dry bean leaves, stems, pods, and seeds were collected from fields located in northeastern North Dakota. These samples were collected during the middle of August when the crop was in mid-seed fill to physiological maturity (growth stages R6 to R8; Osorno et al. 2013; Table 2). Samples with symptoms of anthracnose were obtained from nine fields in Wells County in 2012 (Table 2). In 2014, samples of infected tissue were obtained from a total of 14 dry bean fields in Ramsey, Walsh, Nelson and Traill counties (Table 2).

Table 2. Number and races of Colletotrichum lindemuthianum isolates collected from dry bean fields in North Dakota in 2012 and 2014.

Year collected	County	Field no.	No. of isolates obtained	No. of isolates tested	Races identified
2012	Wells	1	13	3	9, 73(2)
2012	Wells	2	10	3	9, 72, 73
2012	Wells	3	13	3	73
2012	Wells	4	2	2	73
2012	Wells	5	24	3	73
2012	Wells	6	8	3	9, 73(2)
2012	Wells	7	13	3	73
2012	Wells	8	5	3	72, 73(2)
2012	Wells	9	2	2	73
2012	Foster^a	10	8	8	72, 73(7)
2014	Walsh^b	1	5	5	73
2014	Walsh	2	2	2	73
2014	Walsh	3	2	2	73
2014	Walsh	4	4	4	73
2014	Ramsey^b	5	5	5	73
2014	Ramsey	6	5	5	73
2014	Ramsey	7	5	5	73
2014	Ramsey	8	2	2	73
2014	Ramsey	9	5	5	73
2014	Ramsey	10	5	5	73
2014	Ramsey	11	5	5	73
2014	Ramsey	12	5	5	73
2014	Nelson^c	13	1	1	73
2014	Traill^d	14	2	2	73

^aEight C. lindmuthianum isolates were provided by M. Wunsch, NDSU Carrington Research and Extension Center.

^bPlant tissue was provided from Walsh and Ramsey counties by J. Jones, ADM Seedwest.

^cPlant tissue was provided from Nelson County by J. Prischmann, North Dakota State Seed Department.

^dPlant tissue was provided from Traill County by J. Osorno, NDSU Plant Sciences Department.

Pathogen isolation

Dry bean tissue samples were surface-sterilized by soaking them for 1 min in a solution of 10% household bleach (5.25% NaOCl) followed by three rinses in sterile distilled water. The tissue was allowed to hydrate on moist, sterile filter paper for 24 h at room temperature and tissue surrounding a single lesion was excised and crushed. An inoculating loop was used to streak the extract onto acidified potato dextrose agar (APDA) (39 g L⁻¹ distilled water plus 60 µL of 27% lactic acid) with 20 mL 30% streptomycin L⁻¹ water. The plates were incubated for 2–3 days in the dark at 22 ± 2°C. From emerging colonies, the hyphal-tip isolation method was utilized to establish a pure culture on PDA. In addition, eight isolates were provided by Dr Michael Wunsch at the NDSU Research and Extension Center (CREC) in Carrington, North Dakota in 2012.

Inoculum production

Previous research indicated that depending on the race, sporulation of C. lindemuthianum may be sparse and erratic on agar media (Mathur et al. 1950). Therefore, prior to race-typing, a study was performed to determine if media influenced sporulation of C. lindemuthianum. M3 (Pinto et al. 2012) and Mathur’s agar (Balardin et al. 1997) were compared to determine which supported greater sporulation of the pathogen. Agar plugs containing mycelia and spores from actively growing cultures of 39 C. lindemuthianum isolates collected from the 2012 field samples were plated on one Petri plate containing solid M3 or Mathur’s agar and allowed to grow for 7 days in the dark at 22 ± 2°C in a completely randomized design. The agar surface was scraped using 3 mL sterile distilled water and a sterilized bent glass rod. The solution was diluted 1:10 in sterile distilled water, and conidia were counted using a hemocytometer. The experiment was performed twice, and an analysis of variance was conducted on data from the preliminary media using the general linear model procedure in SAS version 9.3 (SAS Institute, Inc., Cary, NC). The number of conidia produced on the two types of media did not differ significantly; therefore, M3 media was used for further experiments (data not shown).

Host inoculation

Isolates of C. lindemuthianum from 2012 and 2014 and a reference culture of race 73, obtained from J. Kelly (Michigan State University), were transferred using agar plugs containing mycelium and spores to M3 medium. The cultures were incubated at 22 ± 2°C in the dark for 7 to 14 days until profuse sporulation was observed on the agar surface.

Seeds of a set of 12 standard host differentials (Table 1) and one known susceptible pinto bean cultivar, ‘Lariat’, were germinated on water agar for one week, and planted in trays (25 cm × 50 cm) containing Sunshine Soil Mix No. 1 (Sun-Gro Horticulture, Canada). Trays were kept in a greenhouse room at 22 ± 2°C with a 14 h photoperiod until the primary leaves were fully expanded (growth stage V1). Three plants per cultivar were inoculated with a suspension containing 10⁶ conidia mL⁻¹ until run-off, placed in a humidity chamber for 5 days at 20°C under 14 h of fluorescent light, and returned to the greenhouse. There were three replications per cultivar, arranged in a completely randomized design. Ten days after inoculation, disease reaction was measured based on a 1–9 severity scale (Balardin et al. 1997). Scores of 1–3 were considered resistant. These plants had no visible disease symptoms or only a few, very small lesions, mostly on the primary leaf veins. Scores of 4–9 were considered susceptible and the plants displayed numerous large lesions or sunken cankers on the abaxial sides of the leaves or hypocotyls (Balardin et al. 1997). Pathogen race designations were determined by adding the binary numbers assigned to each differential that displayed a susceptible reaction (Table 1). Inoculations were performed twice for each isolate.

Results

In 2012, 90 isolates of C. lindemuthianum were recovered from infected plant tissues from nine dry bean fields in Wells County and in 2014, 53 isolates of C. lindemuthianum were recovered from infected plant tissue from 14 fields. No anthracnose was reported in North Dakota in 2013. Of the 33 isolates from 2012 evaluated for race identification, three isolates were determined to be race 9, three isolates were determined to be race 72, and the remaining 27 isolates were determined to be race 73 based on their reactions on the race differential set and compared with the reactions of the isolate previous identified as race 73 (Table 2). All 53 of the 2014 isolates evaluated were determined to be race 73.

Discussion

The results from this study demonstrate that C. lindemuthianum race 73 remains predominant in North Dakota. Diseased pinto bean plants were obtained from nine fields in Wells County and 14 fields in Walsh, Ramsey, Nelson and Traill counties in 2012 and 2014, respectively. ‘Lariat’ was one of the most commonly planted pinto bean cultivars in this area and is known to be susceptible to race 73 (Goswami et al. 2011; Knodel et al. 2014). In 2012 and 2014, there were many nights in which the dew point temperature was reached, making the environment favourable for disease development. The absence of anthracnose in 2013 could have been due to lack of pathogen inoculum or unfavourable environmental conditions during the season. The summer of 2013 was unusually hot and dry, unfavourable conditions for anthracnose development. It is also possible that anthracnose was present in 2013, but those fields were not identified.

Results from inoculation of breeding material from Michigan State with race 73, a Mesoamerican race, indicated that Mesoamerican lines were more susceptible than were Andean breeding lines (Kelly et al. 1994). Additionally, 12 commercial dry bean cultivars (‘T-39’, ‘Matterhorn’, ‘Montcalm’, ‘Avalanche’, ‘Navigator’, ‘Norstar’, ‘Vista’, ‘Lariat’, ‘Maverick’, ‘Othello’, ‘Stampede’ and ‘Sedona’) were evaluated for resistance against previously identified and new races of anthracnose, including 7, 73, 89, 1153 and 1161 (Goswami et al. 2011). Among these cultivars, all except the dark red kidney bean ‘Montcalm’, of Andean origin, were highly susceptible to race 73. Kidney bean acreage in North Dakota during the time period when these isolates were collected was very low (Knodel et al. 2013, 2014), so the resistance offered by ‘Montcalm’ did not likely influence pathogen races. The Mesoamerican differentials ‘Michelite’, ‘Cornell 49242’ and ‘Mexico 222’, which carry resistance genes Co-11, Co-2 and Co-3, respectively, displayed a susceptible phenotype for at least one gene when inoculated with C. lindemuthianum isolates recovered in 2012 and 2014, determined to be races 9, 72 and 73. The reaction on these differentials indicates that these races are of Mesoamerican origin, demonstrating host–pathogen coevolution resulting from selection pressure exerted by the plant and the pathogen (Pastor-Corrales et al. 1995; Young & Kelly 1996; Balardin et al. 1997; Balardin & Kelly 1998; Melotto et al. 2000).

While race 73 continues to be the most common race of C. lindemuthianum identified in North Dakota, other races, including 7, 9, 72, 89, 1153 and 1161, have been found. Races 9 and 72 recovered in the 2012 survey pose no additional threat to the dry bean industry in North Dakota as these races are virulent on resistance genes that are also overcome by race 73. However, races 89, 1153 and 1161 are virulent on additional resistance genes or alleles, but together, they accounted for less than 5% of the total races identified during surveys conducted between 2003 and 2009 (Goswami et al. 2011). The low detection incidence of races 89, 1153 and 1161 may explain why these races were not detected during the survey conducted in 2012 or 2014. Although not detected in North Dakota, race 105 has been reported across the border in Manitoba, Canada. This race has a similar virulence pattern as race 73, except that it is also virulent on ‘Kaboon’ which contains Co-1², of Andean origin. In a previous study, multiple resistance genes effective against races 73 and 105 included Co-1, Co-1⁵, Co-4, Co-4² and Co-5 (Dongfang et al. 2008). Co-4², which encodes a protein kinase (Melotto & Kelly 2001), is of special interest because research has shown that this gene confers resistance to 97% of the C. lindemuthianum races present in North and South America (Balardin & Kelly 1998). Further research is needed to evaluate these genes and others for resistance to the prevalent pathogen races. Few cultivars from the Mesoamerican background with known resistance to race 73 are commonly grown commercially in the USA at this time. Resistance to race 73 is more common in black bean cultivars than pinto, navy and other Mesoamerican types (Kelly et al. 2001, 2005, 2015). Black bean cultivars resistant to race 73 released from the Michigan State breeding programme include ‘Loreto’ (Kelly et al. 2015), ‘Jaguar’ (Kelly et al. 2001), ‘Condor’ (Kelly et al. 2005) and ‘Zenith’ (Kelly et al. 2015). The ongoing evaluation of cultivar responses to pathogen races is paramount to breeding for resistance to C. lindemuthianum and providing growers with resistant cultivars, particularly in the Mesoamerican market classes.

Anthracnose is known to be a seed-transmitted disease; therefore, the use of certified clean seed is one of the most important management tools available to growers. The North Dakota State Seed Department (NDSSD) enforces a zero-tolerance policy to C. lindemuthianum in dry bean seed, i.e. any pathogen detection results in seed-lot rejection (NDSSD 2008). In 2002, the NDSSD began requiring certified seed grown in North Dakota to be tested for anthracnose. In addition, any imported seed is required to be tested for the presence of the pathogen. In 2002, a sample of pink bean seed imported from Manitoba, Canada, was found to be infected with race 1161 (Goswami et al. 2011). This exemplifies the importance of ensuring dry bean seed is tested and certified to be anthracnose-free.

Acknowledgements

This work was supported by North Dakota EPSCoR (NSF Grant number EPS-0814442) and the Northarvest Bean Growers Association. The authors thank Amanda Beck and Inbal Dalit Guinzburg for technical assistance and J. Kelly, Michigan State University, for providing an isolate of C. lindemuthianum race 73. Additionally, we thank M. Wunsch, NDSU Carrington Research and Extension Center, J. Jones, ADM Seedwest, J. Prischmann, North Dakota State Seed Department and J. Osorno, NDSU Plant Sciences Department for providing C. lindemuthianum isolates and anthracnose infected dry bean material.