Simple and rapid detached leaf technique for screening common beans ( Phaseolus vulgarise L . ) in vitro against angular leaf spot ( Pseudocercospora griseola ) disease

1 Southern Agricultural Research Institute, Molecular Biotech Lab., P. O. Box 06 Awassa, Ethiopia. 2 Ethiopian Biotechnology Institute, P. O. Box 32853, Addis Ababa, Ethiopia. 3 Department of Microbial Cellular and Molecular Biology, Addis Ababa University, Ethiopia. 4 International Centre for Tropical Agriculture (CIAT), P. O. Box 6247, Kampala, CIAT-Uganda. 5 Department of Agricultural Resource Management, Embu University College, P.O. Box 6-60100, Embu, Kenya. 6 University of California, Davis Department of Plant Sciences, MS 11 Shields Avenue Davis, California 95616-8780, United States of America.


INTRODUCTION
The angular leaf spot (ALS), caused by the fungus Pseudocercospora griseola, infects common bean (Phaseolus vulgaris L.) throughout its centre of origin and domestication range in the Americas, which extends from the Northwest Argentina to Northern Mexico (Pastor-Corrales et al., 1998).Moreover, this specific disease is *Corresponding author.E-mail: rezene77@gmail.com.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License economically important in Africa (Ddamulira et al., 2015;Olango et al., 2017).It is also a major constraint to common bean production in Ethiopia where this is an important food and economically foreign exchange crop (Lemessa et al., 2011).The ALS disease is favoured by intermittent cool and warm and wet and dry weather (Correa-Victoria et al., 1989).
A precise and reproducible disease screening protocol is important in common bean disease resistance breeding programs to differentiate susceptible and resistant lines in fixed lines and segregating progenies.Screening for ALS resistance may be conducted either in the field or in a screen house; however, both involve tedious procedures and are faced with challenges (Schwartz et al., 1982;Pastor-Corrales et al., 1998;Mahuku et al., 2003;Olango et al., 2017).Field screening is dependent on the natural occurring environmental conditions which are highly unpredictable to cause the right disease pressure in addition to being compromised by the simultaneous occurrence of other bean pathogens.In addition, knowledge of the causative P. griseola pathotype is usually lacking.On the other hand, resistance evaluation under screen house conditions helps to control the unpredictable disease conducive environment and a number of studies have used this method to evaluate common bean genotypes for P. griseola resistance using specific pathogen isolates (Ddamulira et al., 2014;Ddamulira et al., 2015;Olango et al., 2017).However, the achievements of greenhouse screening depend on seedling age, inoculum quality and quantity, inoculation technique, and pre-and postinoculation favourable environmental conditions (Twizeyimana et al., 2007) that are tedious and are prone to human error.The detached leaf disease screening method has been reported as an alternative and rapid technique for screening common bean germplasm for resistance against anthracnose disease (Tu, 1986;Pereira et al., 2013) and whit mold (Teran and Singh, 2009).It was also reported in soybean by Twizeyimana et al. (2007) to screen soybean genotypes for resistance to white-mold disease; the results from the detached leaf assay were reported by the same author and were significantly correlated and comparable with the results of screening house and field screening results.The method has also been reported for screening wheat for fusarium head blight resistance as an alternative disease screening technique (Browne and Cooke, 2004).
Conventional screening methods for common bean (involving attached leaves) against angular leaf spot disease [CIAT screening protocol (Pastor-Carlos et al., 1998)] have been commonly used as an evaluation technique.However, conventional screening techniques have their own drawback for screening common bean for ALS resistance.
The objective of this study was to evaluate an alternative, simple, low cost and high efficiency in vitro screening technique for common bean genotypes against angular leaf spot caused by P. griseola that could be used in common bean breeding programmes.

Description of common bean genotypes
This study was conducted at the Molecular Biotech Lab of the Southern Agriculture Research Institute (SARI), Awassa, Ethiopia.Nine common bean genotypes (Table 1) were selected from a field screening trial during the main growing seasons of 2017 based on their resistance reactions to ALS under natural infection.They included 'kirmson' and G4564 that showed susceptible reaction both in field screening conditions and during the evaluation with the proposed detached-leaf technique.The HSPS10, HSPS11, and HSPS12-genotypes from the present breeding program have a resistant reaction under natural infestation and in vitro with the proposed method.The MEX-54, BAT233 and AND277 are three ALS differentials with known sources of resistance, whereas Redwolaita (RW) is a common bean cultivar known for its susceptibility.

Inoculum preparation and pathogenicity test
Inoculations were conducted with three strains of P. griseola: (1) KA060, received from CIAT-Uganda (Kawanda, Uganda); (2) two pure isolates Pg 001 and Pg 002 that had been previously collected from Wondogenet and Areka bean growing areas of Ethiopia.These isolates were previously characterized for their virulence.Tested strains were grown onto V8 agar medium in the Petri dishes.The resulting spores and mycelia were scrapped smoothly with a spatula and filtered through gauze; the spore concentration was adjusted to 2.0×10 4 conidia/mL using a hemocytometer.
Common bean plants were grown in the screening house.After 18 days of germination, the middle leaflet of the first trifoliate leaf of each plant was detached from each test plant genotype.At this stage, leaves were approximately two-thirds of their full development.The detached leaves set from each genotype were inoculated separately by immersion into the spore suspension and placed in Petri dishes (90 × 15 mm) onto the cotton moistened with 3.0 mL of tap water (Figure 1).The humidity of the Petri dishes was monitored and maintained using applications of watering every three days throughout the incubation period.Dew formed inside the Petri dish allowed the development of the pathogen.The petri dishes were kept at 24°C and incubated for 18 days under a 12-h daily light regime at room temperature.

Determining virulence phenotypes
The virulence analysis was made for each monosporic P. griseola Table 1.Reaction of common bean genotypes under naturally existing angular leaf spot pathogen (Pseudocercospora griseola) at Wondogenet and Areka during the main cropping seasons of 2017 and validation with the proposed method of screening using two identified pathotypes in both location.(1999).The differentials include six large-seeded Andean origin and six small to medium seed sized Middle American origin (Table 3).Three seeds from each differential were grown in plastic pots containing a mixture of soil, sand and organic manure in three replications in the screen house.The plants were inoculated as mentioned earlier.

Disease reaction assessment and data analysis
The treatments were arranged in a randomized complete block design with three replications.Three common bean seedlings per genotype per replication were inoculated as described.The inoculated detached leaves with spore suspension were monitored every day for disease symptom development and the CIAT disease 1 to 9 scoring protocol (Pastor-Carlos et al.,1998) was utilized in which plants that scored 1 to 3 were considered incompatible (-) or resistant and plants scored 4 to 9 were considered as compatible (+) or susceptible.Disease evaluations on individuals on individual detached leaf were made at 10, 12, 14 and 17 days after inoculations, using the 1 to 9 rating scale, where 1=immunity (no visible symptoms) and 9=very severe symptoms (van Schoonhoven and Pastor-Carlos, 1987).

Evaluation of pathogenicity of P. griseola isolates
In vitro (Figure 1) pathogenicity of specific P. griseola isolates, collected from the diverse common bean growing regions of Ethiopia was confirmed by using sets of common bean differentials (Table 3).Hence, the screening technique allowed differentiating between the resistance and susceptible common bean genotypes   (2002) and Mahuku et al. (2004).
(Table 3 and Figure 2).The results from this specific screening technique are comparable and can be used for screening of common bean as resistance/susceptible against leaf spot disease.The backcross progenies with known sources of Phg-2 gene showed higher level of resistance than those without this specific locus (Table 2).All of the susceptible common bean genotypes under the natural infestation during field screening were similarly susceptible and those with high level of resistance were also resistance to the angular leaf spot caused by P. griseola under the rapid screening technique which was used in this specific study.
The procedure is currently part of the common bean breeding program at SARI, Ethiopia and is used in combination with molecular markers to facilitate selection of resistant progenies in each backcross generation.The program adopted this specific technique for screening pyramided back cross progenies with multiple resistance which would have been difficult with the conventional screening technique.Tu (1986) demonstrated many advantages of the detached-leaf screening method of common bean against anthracnose disease as compared to the attached-leaf conventional method of screening.This method can prevent unnecessary interactions between pathogens during bean evaluation for resistance to different pathogens or different races of the same pathogen.With this method of screening, a bean plant can be screened repeatedly because inoculations can be done at any growth stage without affecting the growth and seed production of the rest of the plant.This method also saves time in breeding for resistance and works very well on newly expanding leaves because they appear to have less contamination.This method can speed up selection of progenies during the marker-assisted backcross gene pyramiding and can be used with molecular markers to select progenies with disease resistance traits during each subsequent backcross generations.From the evaluation of a large number of common bean genotypes, four lines of common beans were identified as resistant under field screening at the Wondogenet and Areka research farms during the 2017 cropping season under naturally occurring epidemics.Contrasting pathogen population were also exposed with the proposed screening technique and inoculated with individual pathotype of P. griseola.The genotypes showed comparable reaction with both methods.Hence, the proposed screening technique was found to reliably assess the disease reaction.
In breeding programs and during gene pyramiding, it will also be challenging when using only conventional method of screening to evaluate plant symptoms but this alternative rapid detached leave method is proposed for marker-assisted gene pyramiding in combination with molecular markers to aid the selection of resistant progenies during each backcross generation.In addition, the proposed method can help during evaluation of progenies for different pathotypes using leaves from the same plant simultaneous evaluation from the same plant and this could be difficult with conventional evaluation method (Table 3 and Figure 2).
Finally, the result determined from this specific in vitro screening technique through predetermined pathotype (Ddamulira et al., 2014) which was received from CIAT-Uganda (Table 1) revealed that the proposed procedure and the screening technique was comparable and efficient for pathotype determination using sets of common bean differentials.

Conclusion and implications for common bean breeding
The proposed rapid and efficient in vitro screening technique can be used as an alternative method in common bean breeding programs for screening genotypes for ALS resistance caused by P. griseola.This screening technique can be used in combination with molecular makers to assist and facilitate progeny selection during a marker-assisted gene pyramiding and backcrossing program.The methods can be used during simultaneous evaluation of progenies for multiple resistance as a single plant can be evaluated several times at any growth stage without affecting the growth of the plant, thus, facilitating a selection program at a reduced cost.This alternative screening technique will provide great opportunity for breeders working in gene pyramiding and marker-assisted back-crossing programs to evaluate common bean genotypes and progenies for multiple disease resistance.

Figure 1 .
Figure 1.The proposed rapid screening technique for common bean genotypes against Pseudocercospora griseola, the causal agent of angular leaf spot.a) Single spore isolates.b) Common bean plants growing in the screening house.c) Detached leaf in Petri dish inoculated with single spore isolates and maintained until symptom development.d) Detached leaf in vitro showing ALS symptom development after inoculation.

Figure 2 .
Figure2.Different disease scoring of leaves inoculated with Pseudocercospora griseola, the causal agents of angular leaf spot disease, using the proposed detached-leaf inoculation method. 1 = no visible symptoms; 3 = 10 to 20% of plants infected and/or about 5% of the total plant area affected by the pathogen; 5 = 40 to 50% of plants infected and/or about 20% of plant area affected; 7 = 60 to 70% of plants infected and/or about 40% of plant area affected; and 9 = >80%.Scores 1 -3 considered as a resistance; 4 to 6, moderately resistance; 7 to 9, susceptible.

Table 2 .
Reaction of back cross(BC) segregating progenies for the angular leaf spot resistance.

Table 3 .
Common bean ALS differential cultivar and binary system for pathotype determination study using the proposed rapid technique to confirm predetermined pathotype.