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Characterization and pathogenicity of soil borne pathogens inducing root rot symptoms in common bean in Rwanda

Though Rwanda is among the highest common bean producers and consumers, this crop is still challenged by different factors including root rot diseases resulting in low yield potential. Root rot diseases can cause up to 100% of yield loss. Despite different soil borne pathogens causing bean root rot diseases; there is no recent literature that clearly documents the most aggressive ones affecting common beans in Rwanda. This study aimed at identifying the root rot pathogens affecting bean crop in Rwanda. A survey was conducted to collect bean samples showing root rot symptoms in 12 agro -ecological zones where bean crop is mostly grown. The selection of samples was based on the level of contamination and the purity of cultures. A total number of 132 samples were therefore selected and considered for further analysis. The pathogen isolation was done in Rubona at Rwanda Agriculture and Animal Resources Development Board (RAB) Biotechnology and Pathology laboratory. Structural characterization of soil borne pathogens was also carried out along with pathogenicity evaluation. The growth rate of identified pathogens were significantly different (P<0.001). Significant differences were observed among isolates inoculated on both plantlets and seeds (P<001). Four root rot diseases were identified and characterized including Rhizoctonia spp, Fusarium spp,Sclerotium spp and several Pythium spp. The four pathogens were mostly observed in Northern and Southern parts of Rwanda than East and Western parts of Rwanda. Morphological characterization showed that Fusarium spp, Rhizoctonia spp and Sclerotium spp were predominant than Pythium species. Fusarium spp was identified as the most predominant fungal pathogen while Sclerotium spp was the most virulent pathogen. The present study suggests bean root rot disease control program to reduce losses associated with these diseases especially the use of resistant varieties since the fungi are widespread and survive in soils for a long period of time.


I. Introduction
Common bean (Phaseolus vulgaris L.) is the most important grain legume for direct human consumption in the world. Bean consumption is estimated to more than 300 million people's daily diet [5]. Rwanda is among the highest bean producers and consumers in Sub-Saharan Africa with consumption estimated at200g per capita per day [17]. In some cases, beans are called the meat of the poor due to its high concentration in proteins and its affordability [11], [3] The production in Rwanda is mainly secured by small farmers with average land holdings of less than one hectare and using limited technologies such as limited access to irrigation schemes, limited use of fertilizers and pesticides. Biotic and abiotic factors are the main constraints limiting common bean productivity in Sub-Saharan African countries [2] [17] [17]. Root rot disease caused by soil borne pathogens is among the biotic constraints affecting bean productivity. A number of these soil borne pathogens has been identified in Latin America and Africa such as Fusarium solani f.sp. Phaseoli, Pythium spp, Rhizoctonia solani, Macrophomina phaseoli and Sclerotium rolfsii [6] Root rot diseases are most damaging when farming conditions are poor; including water logging, poor drainage capacity, intensification of the cultivation of beans, decline in soil fertility with absence of rotation and use of susceptible varieties [14]. The bean production system in Rwanda is characterized by series of different specific conditions where common bean varieties are grown under high demographic pressure, poor soils with very limited use of crop rotation [10] [18].
Symptoms and severity of root rot diseases depend on the environment conditions including rainfall, moisture, temperature, soil type, drainage, cropping system, and the interaction between responsible pathogens. The objective of this study was to identify and characterize root rot diseases caused by soil borne pathogens of common bean (Phaseolus vulgaris) in Rwanda.

II. Materials and methods 2.1. The study location and sampling methodology
The study was conducted in all 12agro-ecological zones of Rwanda where bean is grown. Samples were collected in common bean fields showing above ground root rot symptoms such as low plant vigor, yellowing and stunting. A total of 450 bean fields were visited. Three sectors were selected in each district and five bean fields in each sector were surveyed, considering the distance of 2km from one field to another. In each bean field, five quadrants each measuring 1m 2 were randomly designed and bean plants showing root rot symptoms were collected from them. A total of 2118 bean samples were collected and samples that did not show any growth of mycelia were discarded along with samples that showed visual contamination with nontargeted pathogens. The disease plant was completely uprooted and put in labeled paper bags. In each sampled field, administrative and geographical information (district, sector, cell, village, altitude, and longitude, latitude, cropping system and soil type) were recorded. One hundred and thirty two samples were then brought to the laboratory for pathogen isolation.

Preparation of the media
Different types of culture media such as Potato Dextrose Agar (PDA), Corn Meal Agar (CMA) and Tap Water Agar (TWA) were used for fungal isolation and growth.

Preparation of tap water agar
Sixteen grams of agar extra-pure were added in 1000ml of tap water, shaken until the solution reaches the homogeneity stage and autoclaved at 121 o C for 20 minutes. The autoclaved media was allowed to cool down to 40 o Cunder laminar flow then dispensed in 9 mm wide petri dishes in the rate of 20 ml per petri dish. The collected samples were prepared into piece of roots, isolated on tap water agar and incubated at 23 o C for 24 hours. Pathogen isolation was performed using a laboratory protocol as described in the International Center for Tropical Agriculture (CIAT) training manual (Mukamuhirwa et al., 2017). All emerging fungi from the roots cultured on tap water agar were sub cultured on PDA and CMA.

Preparation of Potato dextrose Agar and Corn Meal Agar
Sixteen grams of PDA or CMA were added to 1000 ml of distilled sterilized water. The mixtures were shaken until the solutions reach the homogeneity stage and autoclaved at 121 o C for 20 minutes. The autoclaved media was allowed to cool down to 40 o C under laminar flow then dispensed in 9 mm wide petri dishes in the rate of 20 ml per petri dish. A bock agar from tap water media was transferred to the petri dishes with PDA to identify Rhizoctonia spp, Sclerotium spp and Fusarium spp while CMA was used to identify Pithium spp from other root rot pathogens. The petri dishes were incubated at 23 o C [14].

Pathogens isolation
The pathogen isolation was carried out in the Rwanda Agriculture and Animal Resources Development Board (RAB), Biotechnology and Pathology Laboratories. A single hyphal isolation was conducted under microscope on mycelium tips. All isolates were transferred on fresh medium of PDA and CMA with sterilized filter paper (Waltman paper n o ) for root rot pathogens long term conservation. Filter paper was cut into small pieces of about 4cm squared. The pieces of paper were then folded into aluminium paper and sterilized at 121 0 C for 20 minutes. The pieces of paper were cooled down to room temperature. Using sterilized forceps under laminar flow, about 20 sterilized pieces of filter papers were placed on PDA and/ or CMA containing colonized agar block to allow the pathogens to grow and colonize the filter papers. Colonized filter papers were then transferred to new sterilized empty petri dishes. These petri dishes were placed into sterilized desiccators for two to three days to allow the filter papers to dry. The dried colonized filter papers were removed and wrapped in sterile Aluminum foil, clearly labeled and stored under -20 o C in freezer for further use.

Pathogens isolation
The pathogen isolation was carried out in the Rwanda Agriculture and Animal Resources Development Board (RAB), Biotechnology and Pathology Laboratories. A single hyphal isolation was conducted under microscope on mycelium tips. All isolates were transferred on fresh medium of PDA and CMA with sterilized filter paper (Waltman paper n o ) for root rot pathogens long term conservation. Filter paper was cut into small pieces of about 4cm squared. The pieces of paper were then folded into aluminium paper and sterilized at 121 0 C for 20 minutes. The pieces of paper were cooled down to room temperature. Using sterilized forceps under laminar flow, about 20 sterilized pieces of filter papers were placed on PDA and/ or CMA containing colonized agar block to allow the pathogens to grow and colonize the filter papers. Colonized filter papers were then transferred to new sterilized empty petri dishes. These petri dishes were placed into sterilized desiccators for two to three days to allow the filter papers to dry. The dried colonized filter papers were removed and wrapped in sterile Aluminum foil, clearly labeled and stored under -20 o C in freezer for further use

Structural characterization of soil borne
pathogens Cultures were initiated for the growth rate measurements and phenotypic description of different pathogens. The description of the different root rot pathogens was done using their colony texture, mycelia color, presence or absence of sclerotia, color of sclerotia, colony margin and number of sclerotia, clear zonation and mycelia floral patterns. Microscopic identification of the pathogens was also done basing on the presence of micro and macro conidia (Fusarium spp.), and the hyphal branching at the perpendicular angles or Tshaped branching (Rhizoctonia spp.). Colony diameters for all cultures initiated for growth rate variations were measured after 24 hours of incubation. This was done by drawing two perpendicular lines at the base of the petri-dish and colony margins measured using a ruler along the lines. Measurements were carried on every day until when the plates were completely covered.

Pathogenicity evaluation
The pathogenicity test of isolated pathogens was performed using 132 isolates. The sorghum grain media was used for fungal inoculum production. Approximately 400ml of distilled water were added to every 300g of sorghum grains, loaded into a sterilized glass bottle, the autoclaved twice at 121 o C for 20min and allowed to cool for 12 hours. The sterilized sorghum was then inoculated with the liquid suspension of 100 μl scraped pathogen mycelia dissolved in sterile distilled water. The media substrate bottles were hand shaken daily to homogenize the fungal distribution and growth. The inoculum was incubated in the bottles over the sterilized sorghum grains in a sterile environment at room temperature for 14 days to allow uniform growth [4] [7]. Pathogenicity evaluation was done through 3 different experiments while the seed rot experiment was conducted in petri dishes following the MSU protocol where the colonized agar block was placed in the middle of fresh media and allowed to grow and infect the 10 sterilized healthy seed in the petri dish. The incubation was done for 7 days and the disease severity was assessed using 1-5 scale where 1: Germinated, 2: Delayed germination; 3: Germination and some Lesions; 4: Germination with coalesced lesions; 5: Seed colonized. The second experiment was conducted on laboratory bench where plastic boxes where used to plant sterilized healthy seed on sterilized paper towel. Bean seed were allowed to grow for 2 weeks, taking care of removal of any plant showing the symptoms of the disease. This was followed by inoculation of roots of plantlets with colonized sorghum grain, incubate for 7 days as explained above and evaluate the severity of the disease using 1-5CIAT scale. The third experiment was set in pots of 5liters in screen house following CIAT Training manual.

Statistical analysis
The slope between the first and last day of measurement was subjected to Analysis of Variance (ANOVA). The Disease Index (DI) scored on seed rot experiment was calculated as: DI = Σ(severity X n) / N [8] and the means were alsosubjected to ANOVA. Disease severity for plantlet inoculation in laboratory bench and screen house data in pots were recorded following CIAT scale and subjected to ANOVA.All the analysis were performed using Gen-stat Statistical Package.

3.1.
Morphological characteristics and distribution of root rot diseases in Rwanda The colony type of identified isolates was 69 % cotton and 31% flat. The regular and or round colony margin was observed at 43 % while the irregular colony margin was observed at 57%. The mycelia of the identified pathogens were raised at 70% and thin at 30%. From the identified pathogens the mycelia color was 36% purple, 14% red, 25% cream and 25 % white. The growth rate of identified pathogens were significantly different (P<0.001) with the range of slope varying between 0.1 to 5.3 mm (Table 1). How many isolates were classified as fast growing, slow or medium?Among the 132 isolates, 68%, 9%, 32%, and 30% wereFusarium spp, Pythium spp, Rhizoctonia spp, and Sclerotium spp respectively. Morphological characterization showed that Fusarium, Rhizoctonia and Sclerotium isolates were more predominant than Pythium species (Figure 1

2. Pathogenic characterization
The most virulent isolates were selected for screening varieties for resistance to root rot pathogens. Selection of virulent isolate was done in 3 experiments which showed the same results. The study identified a high level of diversity in bean root rot diseases between (Figure 3:F1) and within species (Figure 3:F2 and F3). The four diseased were observed especially in Figure 1. The virulence study showed that isolates from southern and western locations were more virulent than the rest.  Figure  2). The Greenhouse experiment confirmed the results of seed rot assay and plantlet rot experiments ( Table 3). The most aggressive isolates selected based on all experiments. Figure 2: Map of the study area    In the present study, diseased locations were observed especially in northern and southern parts of Rwanda while east and western parts were less affected. Several studies showed that the distribution of bean root rot diseases depend on several factors including species type [14]. The most virulent pathogens were found within Sclerotium isolates and it was scored high compared to other pathogens. This fungus can infect any part of the plant and the variability among isolates is well documented. The aggressiveness depends on the environment interaction among other factors.

V. Conclusion and recommendations
Four root rot diseases were identified and characterized including Rhizoctoniaspp, Fusariumspp, Sclerotiumspp and several Pythium spp. Morphological characterization showed that Fusarium spp, Rhizoctonia spp and Sclerotium spp were predominant than Pythium species. The four diseased were observed especially in northern and southern parts of Rwanda than east and western parts of Rwanda. The collected and characterized isolates constitute an important resource for screening and improvement of dry bean germoplasm in Rwanda. Fusarium was identified as the most predominant fungal pathogen in Rwanda. It was observed that Sclerotium was the most virulent pathogens since most of its isolates scored high compared to other pathogens. The selected isolates are highly recommended to be used in selecting breeding materials in Rwanda. The total bean root rot disease control program is recommended to reduce losses associated with these diseases especially the use of resistant varieties since the fungi are widespread and survive in soils for a long time. It is highly recommended to promote the use of integrated technologies to reduce bean root rot causing pathogens especially Sclerotium. Further studies are recommended to characterize the identified pathogens.

VI. Acknowledgement
The author would like to acknowledge the contribution of Fred Kato from CIAT Uganda, Annuarite Uwera, Vedaste Ahishakiye, Nyirahategekima Esperance, the bean team of RAB, laboratory staff of RAB and all other scientists who contributed to this work. We are grateful to Rwanda Agriculture Board, CIAT Africa, NIFA project for all the support to this work.