Multiple genes confer anthracnose resistance in French bean accessions of Garhwal Himalayas

The Indian Himalayan region is very rich in the genetic variability of French bean and therefore considered as the secondary centre of origin of French bean. Though, a good diversity of French bean is present in Uttarakhand hills of Western Himalaya but it is almost unexplored yet. Unfortunately, French bean is attacked by some of the phytopathogens those cause heavy crop losses. Colletotrichum lindemuthianu (Sacc. & Magnus) Scrib is one of the sever pathogens that causes anthracnose disease in French bean worldwide. Identication or development of a resistant variety/cultivar is an environmentally safe approach. Diversity analysis of anthracnose resistant loci in common bean is important to identify new sources of resistance gene(s). So the study was designed with the objectives to collect and screen the local accessions of French bean from Garhwal hills for anthracnose resistance. A total of 100 accessions were collected from 6 different districts of Garhwal region of Uttarakhand and all were screened for anthracnose resistance. For this, 13 SCAR (Sequence cleaved amplied region) primers specic to anthracnose resistance were used in this study. Results revealed a high level of genetic diversity within the population for anthracnose resistance loci. Gene diversity ranged from 0.354 to 0.499 with a mean of 0.457. Pair wise genetic distance ranged from 0 to 2.236. The accessions were also screened for anthracnose resistance under eld and polyhouse conditions. There was a moderate correlation (R = 0.56) between eld trial and trial under controlled condition. Thirteen of these accessions possessing two genes (Co-10 and Co-42) showed complete resistance for anthracnose disease under eld and polyhouse conditions. The anthracnose resistant accessions may further be used in future breeding programmes to develop new and more resistant varieties of French bean against anthracnose disease.

In Uttarakhand the cultivation of French bean is mainly done by small to medium scale farmers and the enterprise creates on farm employment opportunities for the rural community. However, the disease alone accounts for around 50 % losses in yield and also reduces quality of the produce (Sharma and Sugha 1995). Fungus requires cool and humid conditions to grow and the environmental conditions of Uttarakhand hills favor the development of this disease. Because of its seed borne nature and pathogenic variability disease can cause rigorous yield loss. The pathogen's reproduction mechanism is responsible for continuous resistance development, which is mainly observed in commercial cultivars, since most of them have vertical resistance that is easily overcame by emerging races of this fungus (Rodriguez- Guerra et al. 2003). However, among the various strategies adopted to control anthracnose disease, use of disease resistant varieties is considered as the most e cient and economical method (Mahuku et al. 2002;Pastor-Corrales et al. 1995;Schwartz et al. 1982).
Currently, twenty-one anthracnose resistance loci have been characterized and are denoted by the symbol Co followed by a numerical designation (Kelly and Vallejo 2004). In addition to these, four allelic series have also been identi ed to contribute is resistance in French bean against C. lindemuthianum. However, different varieties/ accessions of French bean possess variable resistance loci to confer disease resistance (Ferreira et al. 2013). Though, a large number of literature on research on anthracnose resistance in French bean is available, but, still bean breeders are putting great efforts to assess the speci c gene(s), which should be deployed in resistance breeding programmes. In addition to identi cation of resistance by conventional methods, the use of molecular markers has contributed signi cantly to characterize resistant genes for anthracnose disease. Use of molecular markers for screening of germpalsm reduces the time and costs involved in the whole process. Since DNA markers are closely linked to genes and are not in uenced by environmental factors, they show epistatic or minimum/none pleiotropic effects (Agrawal et al. 2008).
Among the available molecular markers, the SCAR (Sequence characterized ampli ed regions) markers have been playing great importance on common bean analyses for anthracnose resistance. These markers have been optimized in breeding programs dedicated to search anthracnose resistant cultivars by implanting assisted backcrosses (Miklas and Kelly 2002), during characterization of accessions in the beginning of the selection process or to obtain superior lineages (Beraldo et al. 2009). Until now, there are 14 SCAR markers have been linked to anthracnose resistance genes.
Wide but unexplored genetic diversity of French bean is found in the Uttarakhand hills that has noticeable variation particularly in regards to the edible parts and growing habits. Unfortunately, much effort has not been made to characterize and explore this diverse gene pool of French bean. There is a need for more inclusive analysis of genetic diversity and resistance for different diseases of germplasm available in this region. Therefore, the present work was aimed to identify anthracnose resistant accessions of French bean through the evaluation of germplasm collected from Garhwal Himalayas. Efforts are also done to identify the presence of resistance loci and their contribution in asserting the resistance against anthracnose disease using SCAR markers.

Seed Material
One hundred accessions of French bean were collected from the different locations of six districts of Uttarakhand, India. However, a mixed germplasm of French bean was obtained from the farmers of most of the locations. The germplasm was therefore physically puri ed on the basis of seed colour, size, shape and texture etc. Accessions were then multiplied at the research block of Dept. of Seed Science and Technology, HNB Garhwal University to assess the presence of anthracnose resistance gene(s) by molecular and morphological markers. Two anthracnose resistant lines D line (Cornell-49242) and L line (G-2333) were procured from Dr. P. N. Sharma, Head, Dept of Plant Pathology, CSK Himachal Pradesh Krishi Vishwavidyalaya, Palampur, Himachal Pradesh and used as control in this study.

Molecular Screening
Thirteen SCAR primers speci c to anthracnose resistance and covering all the chromosomes of French bean were used for screening of germplasm (Table 1). Genomic DNA was isolated by cetyl trimethyl-ammonium bromide (CTAB) method as described by Doyle and Doyle (1990). Reaction mixture (10µl ) for the ampli cation was prepared containing 50ng DNA, 2X PCR Buffer, 1µM primer, 100 µM of each dNTP, 0.3 U Taq The primers received from the manufacturer were diluted to prepare working stock solution of 10µM concentration. However, for ampli cation of the DNA, 1µM of the speci c primer(s) was used.
Screening under eld condition

Pathogenic inoculum
A pre-identi ed virulent strain C. lindemuthianum DY-27 was obtained from well characterized repository of Microbiology Lab, Dept. of Basic Sciences, College of Forestry, Ranichauri and used to study the resistance potential of French bean accessions. The fungal strain was grown on potato dextrose agar (PDA) plates amended with streptopenicillin (Sarabhai Zydus Pvt. Ltd., India). The plates were then incubated at 25 ± 2°C for 7 days. Conidia were scraped from incubated plates in to 10-20 ml of sterilized distilled water, and nal volume was made up to 50 ml with sterile distilled water. Spore suspension was ltered through sterile muslin cloth, and spore concentration was adjusted to 5×10 5 ml − 1 . Three to four drops of Tween-20 (0.01 %) were added to it just before spraying.

Pathogen inoculation
Twenty-one-day old plants were used for the infection assay. Three seedlings per pot for each accession were maintained. The experiment was conducted in triplicate under polyhouse conditions. Seedlings were then sprayed uniformly with spore suspension of C. lindemuthianum DY-27. Humidity in the polyhouse was maintained by regular sprays of water with overhead sprinklers. Disease reactions were scored visually after 7 days of inoculation on a scale Genetic correlations between both the disease environments ( eld and controlled conditions) were estimated from predicted disease reaction values in both the conditions, using the Pearson correlation coe cients (Steel et al. 1997).
The K-means algorithm was used to make clusters on the basis of disease reaction under eld and in-vitro conditions. The objective of using non-hierarchical cluster function is to minimize the sum of the squared distances of accessions from their cluster.
Therefore, we did not consider this primer to portray the results. For rest of the primers, the ampli ed bands ranged from 360 bp to 1076 bp ( Fig. 1a, Table 2). The Co-6 gene was present in 62 accessions followed by Co-10 (52) and Co-3/Co-9 (37) ( Table 2). Results also revealed the presence of Co-10 gene in all resistant accessions, followed by Co-4 2 (92.31%) accessions and Co-6 (84.62%) (Fig. 1d).   Table 1). The moderately resistant (21) and resistant accessions (31) were further screened under polyhouse conditions through arti cial inoculation. Under polyhouse condition out of 52 accessions (resistant and moderately resistant), 13 accessions were found resistant, 32 were moderately resistant and 7 were susceptible for anthracnose disease (Supplemental Table 2  showed resistance towards anthracnose under eld conditions but on arti cial inoculation they showed vulnerability towards anthracnose disease (Supplemental Table 2 (Table 4).  In the present study, SCAR markers were successfully used to assess the genetic diversity of anthracnose resistant loci among 100 common bean genotypes collected from Garhwal hills. These SCAR primers were speci cally selected to cover all the linkage groups. Our results were close to some earlier studies carried out on common beans by other researchers. Mean PIC, GD and frequency of allele were 0.452, 0.457 and 0.55 respectively ( Table 2). The ndings suggest that the bean loci identi ed by SCAR markers have moderate level of genetic diversity. The moderate level of heterozygosity observed in these accessions might be due to predominantly autogamous habit of common bean. There are several hypotheses which explain such intra-accession variation, such as, the mixed-mating reproductive system of bean, with up to 10% outcrossing ( However, it was observed that the accessions having Co-10 and Co-4 2 gene together were found resistant for anthracnose. All the 13 accessions those were found resistant under eld and polyhouse conditions possess these two genes consistently with some other genes. However, GFB-4 was found as an exception to this as it contains both the genes eventhough it is moderately resistant for anthracnose (Supplemental Table 1, 2). While accessions GFB-75 and GFB-76 were having only these two genes and were found resistant for anthracnose (Supplemental Table 1). It clearly suggests that both the genes together confer resistance for anthracnose in Garhwal Himalayan region. Banoo et al.
(2020) collected 188 common bean landraces from North-West region in India and screened against ve important anthracnose races. They found that the presence of Co-4 and Co-2 genes in common bean landraces was encouraging for breeding durable anthracnose resistant cultivars for the region. French beans from Harshil and Chakarata are preferred by the consumers for their taste, cooking quality and digestibility. However, the accessions from Harshil were found tolerant while from Chakrata beans were susceptible to anthracnose disease (Supplemental Table 1). It is therefore necessary to identify or develop anthracnose resistant accessions for these regions so that the economic bene t of the farmers could increase.
Correlation coe cient is important in plant breeding because it measures the degree of association between two or more factors (biotic and abiotic) (Dewey and Lu 1959). It is clearly visible in Fig. 4 where trend line showing more resistant accessions when screened under eld condition. Results of controlled condition inoculation and natural led infection were not highly correlated in this study (Table 4). Some accessions which were resistant under eld conditions sowed susceptibility for anthracnose under polyhouse trial (Supplemental Table 2). This greater experimental accuracy is mainly due to su cient amount of pathogen inoculums, controlled temperature and humidity in polyhouse, providing favorable environment for progress of disease. For disease screening of germplasm it is necessary to screen germplasm under in-vitro conditions. Our ndings get support from Leite et al. (2016), who reported resistance in common bean progenies for white mold (Sclerotinia sclerotiorum Lib.) in eld and greenhouse experiments. To determine the spectrum of resistance in the 14 bean genotypes, seedlings were inoculated with anthracnose inoculums under controlled condition (Gonc¸alves-Vidigal et al. 2020). The advantage of in-vitro screening methods is that the most favourable conditions for disease progression can be provided. So, if the cultivar is identi ed as resistant to disease, the cultivar can further be considered for breeding programmes. In addition, these methods reduce the risk of the pathogen spreading to the surrounding environments.
A large number of Co-genes have been substantiated in the anthracnose differential cultivars and accessions (Melotto et al. 2000). These genes are presented as part of a multi-allelic series or in group with other Co genes. They are most likely subsisting resistance gene clusters and have been established at the molecular level for the B4 R-gene cluster for anthracnose resistance. This is well known that the resistance provided by a single gene gets easily broken down. It is therefore needed to pyramid the genes for anthracnose resistance in case of French bean too. This is also suggested that while selecting for anthracnose resistance in a particular region, bean breeders should carefully choose a gene pair that, if deployed singly, would confer resistance to all known races in that region. New resistance source for anthracnose needs to be identi ed to expand the resistance spectrum of future bean cultivars.
Genes Co-10 and Co-4 2 together were found able to provide maximum resistance against anthracnose disease in French bean accessions collected from Garhwal Himalayan regions. Information generated during this study may be helpful in getting information about the resistance level of accessions from different regions. The identi cation of agronomically superior and anthracnose resistant accessions will be useful in minimizing the linkage drag usually breeders come across while transferring disease resistance in already available high yielding but susceptible varieties.
Further, subsequent analysis of diversity using more speci c molecular markers is required to explicate more information on the overall genetic diversity, origin and particular gene(s) responsible for resistance in the Himalayan gene pool.

Con ict of interest statement
The authors declare that the research was conducted in the absence of any commercial or nancial relationships that could be construed as a potential con ict of interest.

DATA availability
Data generated during this study are included in this published article and its supplemental les. Requests for additional information regarding the elite genetic materials in this study can be made to the authors and will be considered without undue reservation. References