Morphological and molecular identification of Pythium spp . isolated from common beans ( Phaseolus vulgaris ) infected with root rot disease

Common beans (Phaseolus vulgaris L.) is the main leguminous crop grown primarily by small-holder farmers in the East and South African countries. Pythium root rot disease is the major production constraints which results in yield losses of 70% to most commercial bean cultivars in eastern Africa. Study focused on ascertaining preliminary information on bean cultivation practices in Tanzania, morphological and molecular characterization and identification of Pythium species from infected beans plants and determining the relationship between soil pH and the occurrence and distribution of the Pythium spp. Soil samples and infected bean plants were collected by aseptic pathogenic isolation and DNA extraction. Universal primers (ITS1 and ITS4) were used for amplification and followed by sequencing. About 63.0% of farmers practiced sole beans cropping, 31.0% mixed cropping and 6.0% intercropping. Corn, banana, cassava, Irish potatoes and coffee were either mixed or intercropped with beans. Also, 52.4% of farmers use farm saved seeds and 92.9% do not use fertilizer in their bean fields. Eleven species of the Pythium spp. were identified: Pythium aphanidermatum, Pythium splendens, Pythium ultimum, Pythium attrantheridium, Pythium graminicola, Pythium oligandrum, Pythium dissotocum, Pythium irregurale, Pythium camurandrum, Pythium paroecandrum and Pythium acanthophoron. Phylogenetic analysis showed diversity and homogenity among the Pythium spp. across the collection area. A high incidence and wide distribution of Pythium species were recorded in soils in the 5.03 to 5.95 pH range.

well as for cash earnings (Hillocks et al., 2006).This region is the most important common bean production area in sub-Saharan Africa and has a high varietal diversity of the crop (Fivawo and Msolla, 2011).Production of common beans in different production areas is hampered by various biotic and abiotic factors which lead to continuous decline of the crop production per unit area (Hillocks et al., 2006).Soil borne diseases caused by either Fusarium spp., Pythium spp.and or Rhizoctonia spp.are biotic constraints to the production of common beans in East African regions; these pathogens act either individually or in a complex manner (Rusuku et al., 1997).Root rot diseases have received little attention until recent years when they became major concerns in East Africa (CIAT, 2003).In Tanzania, limited studies have been conducted regarding this disease and its causative microorganisms.Cultivation of most of the popular commercial common bean genotypes in parts of East Africa is constrained by Pythium root rot which results in yield losses of up to 70%.Predictive models have been used to identify new areas where root rots are expected to become a serious problem in Tanzania (Morogoro, Usambara Mountains, parts of Kilimanjaro, Arusha, Mbeya and Kagera), Kenya (Kisii and Nyahururu) and Uganda (Nebi, Apac and parts of Ntungamo) where farmers have already started to encounter root rots, Malawi (the Chitipa Highlands and Shire Highlands), Mozambique (Manica and Lichinga) and Ethiopia (Hararghe) (CIAT, 2003;Wortmann et al., 1998).Therefore, this study aimed at i) ascertaining preliminary information on bean cultivation practices in bean growing parts in Tanzania, ii) in-depth scientific investigation on the incidence and occurrence of Pythium root rot disease in relationship with soil pH within some parts of Tanzania.

Collection of diseased plants and soil samples
From surveyed farmers' fields, six infected bean plants showing symptoms of root rot disease were characterized by poor seedling establishment, damping-off, stunting and premature defoliation, deterioration of leaves, plant wilt and death.Symptomatic plants were uprooted using a shovel (Mwang'ombe et al., 2007), put in paper bags and transported to Sokoine University of Agriculture (SUA) Laboratories, Tanzania.Soil samples were also collected at random in the farm at a depth of 0-10 cm with soil auger.Soil pH was determined in a ratio of 1:2.5 soil : water suspension by the potentiometric method (McLean, 1982).

Isolation of Pythium spp. from infected bean plants
Corn meal agar (CMA) growth media (17 g in 1000 ml of distilled water) was autoclaved at 121°C for 15 minu; when media was cooled at 40°C, 3 and 0.15 mL of the antibiotics Rifampicin and Pimaricin were added, respectively.Approximately 0.5 to 2 cm of infected root tissue was cut and rinsed first in 70% ethanol for 30 s then in 2% solution of sodium hypochlorite (NaClO) for 1-2 min and finally rinsed twice in sterilized distilled water.Cut tissues were blotted dry on sterile filter paper, plated on CMA growth media and incubated for 5-7 days at 24°C to allow growth of the pathogen.Based on morphological characteristics of sporangia, oogonia wall, antheridia and oospores are distinctive features of Pythium from other root rot pathogens.Purification of Pythium culture was carried out by cutting a small piece of the media with mycelia from the edge of a colony and then subcultured onto new growth media.Pure isolates were transferred to potato dextrose agar (PDA) slants and after 14 days were stored at -20°C.

DNA extraction
Prior to extraction, pure isolates of Pythium were reactivated by sub-culturing on PDA growth media and incubated at 24°C for 14 days to allow massive production of mycelia.DNA was extracted from mycelia of Pythium using the protocol developed by Mahuku (2004).

Polymerase chain reaction (PCR)
The internal transcribed sequence (ITS) region was amplified using universal primers ITS1 and ITS4.A reaction volume of 50 μL containing 23.0µL nuclease free water, 25.0 μL of EconoTaqPLUS GREEN 2X Master, 0.5 µl of each primer (10µM) [ITS1 (5'-TCC GTA GGT GAA CCT GCG G-3') and ITS4 (5'-TCC TCC GCT TAT TGA TAT GC-3')] and 1.0 μL of DNA template (Lucigen Corporation 2505 Parmenter St, Middleton, WI 53562 USA) was used.Amplification conditions were achieved in a BIO RAD My Cycler thermal cycler programmed for initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 57°C for 30 s and extension at 72°C for 1 min.At the end of amplification reaction, a final extension step was accomplished at 72°C for 10 min.PCR products attained were run at 1% agarose gels dissolved in 1× TAE (Tris-Acetate EDTA buffer) concentration as the running solution followed with post staining of ethidium bromide (0.5 µg/ml).Electrophoretic migration was carried out for 1 h electrophoresed at 100 V.The amplified products were visualized and photographed under ultraviolet (UV) light.A 100 bp EZ Load molecular ruler (Bio-Rad Laboratories, Inc. CA, USA) was used to estimate the size of PCR products.

Sequencing, identification and phylogenetic analysis
PCR products with a size of 450 bp and above were sent to Beckman Coulter Company and 43 PCR products were subjected to single pass sequencing (Beckman Coulter Genomics, Inc. Danvers, MA USA).ITS sequences of Pythium isolates were compared with ITS sequences of known Pythium species available in the GenBank database by performing nucleotide blast search at the National Center for Biotechnology Information (NCBI) website (http://blast.ncbi.nlm.nih.gov/blast.cgi).The MEGA 6 software was used for phylogenetic analysis (Tamura et al., 2013).

Statistical analysis
The data collected from the survey was summarized using descriptive statistics such as means, frequencies, percentages and cross tabulations were used to establish the strength of association  between variables (SPSS Inc., Chertsey, England) (Steel et al., 1997).

Root rot distribution
Root rot disease severity was identified from selected hot spot areas, using 84 samples collected which include 43 samples from Lushoto district (five ward locations; Lushoto, Ubiri, Lukozi, Gare and Kwemashai) and 41 samples from Mbozi district (four ward locations; Ruanda, Igamba, Mlowo and Myovizi).

Production practices of common beans producers within the sampling area
Most of the farmers (63.0%) practiced sole cropping of beans, 31.0%mixed cropping and 6.0% intercropping.1).

Occurrence of root rot symptoms in farmers' field
Deterioration of the leaves, wilting, water soaked roots and spongy with discolored cavities dominated in several fields at a frequency of 40.4%, dropping of yellow leaves, stunted growth, uneven growth and poor germination was reported in 35.8% of samples, death of roots and emergence of adventitious roots above the dead root parts and extended brownish color in 19.0% of the samples, water soaked stem extended to hypocotyl of bean seedlings in 4.8% of the samples (Table 2).About 50.0% of respondents indicated that they had observed the above symptoms in their fields in all the seasons, 35.7% observed these symptoms before the 2012 seasons, 8.3% during the 2013 season, and 6.0% throughout the 2014 season (Table 3).

Relationship of Soil pH and disease occurrence
The pH of soil samples collected in the study areas ranged between 5.03 -6.41 of which 23 isolates were found in pH range of 5.03 -5.95 and 9 isolates in soil pH range of 6.05 -6.41.None of the soils had pH higher than 6.5 indicating that, these soils are acidic.Though, P. splendens and P. aphanidermatum were found in both low and higher ends of the acidic spectrum, P. splendens was found in most soils with lower acidic pH values of between 5.03 -6.12; while P. aphanidermatum was found in mostly soils with higher pH values of between 5.41 -6.41 (Table 4).

Phylogenetic relationship of Pythium spp.
Five clusters with ten sub-clusters showed diversity and homogenity of species within geographical location (Figure 3).Cluster specific sequences were dispersed over the ITS regions and contributed to the divergence between clusters and convergence between sub-clusters.In cluster I, convergence was observed between P. aphanidermatum and other species, in particular P. dissotocum, P. acanthophoron and P. oligandrum although they originated from different geographical locations.In cluster II, P. attrantheridium, P. paroecandrum, P. irregurale and P. graminicola were clustered together due to their close relationships.P. splendens and P. aphanidermatum in cluster III were closely related despite their origin.Likewise, alignments revealed that, P. aphanidermatum, P. ultimum and P. oligandrum were closely clustered together due to their similar origin in Lushoto district under cluster IV.P. camurandrum is the only isolate having distant relationship from other Pythium species.

DISCUSSION
According to the survey conducted in this study, similar root rot symptoms were observed as described by previous findings (Abawi et al., 2006;Agrios, 2005;Buruchara et al., 2010).More farmers use farm saved seeds in which survival structures of the pathogen can be stored together with the seeds and when planted they germinate together and lead to infection and development of the disease.Seed rot and preemergence damping-off normally reduce germination rates of planted cultivars due to infection caused by Pythium species (Xi et al., 1995).Sole cropping of bean was found to be the dominant system of cultivation as compared to mixed cropping and intercropping due to short rainy season.Previous studies in Uganda showed the incidence of Pythium root rot disease in mixed cropping systems.Potatoes, sorghum, maize and peas were found to be susceptible and infected by Pythium when intercropped with beans and the cultivation of common beans in mixed cropping systems with other crop species.This partly contributed to beans root rot epidemics and sorghum and peas were found to be the alternative hosts of the pathogenic Pythium species (Gichuru, 2008).Studies conducted in Japan showed prevalence of P. paroecandrum, P. spinosum and P. ultimum infecting common beans in rotation plots rather than in sole bean cropping plots because of inoculum build up from other alternate crop prone to root rot (Kageyama, 1981).Soil pH influences some life cycle stages of Pythium species particularly during formation of oospores and sporangia (Martin and Loper, 1999).This study showed that more incidences of Pythium pathogen were found between soil pH of 5.1 to 5.6 and less incidence occurred between 6.1 to 6.5 pH; these soils are classified as strongly/moderately acid and slightly acidic, respectively (Soil Survey staff, 1993).Soil pH affects composition of the root exudates, which attract soil borne pathogens (Agrios, 2005).The use of liming materials balances the soil pH to neutrality, and the response of liming materials to soil pH increase is because it removes imbalance of nutrients particularly reduction of aluminum and   manganese toxicities which provides calcium ions to counteract its deficiency (Biswas and Mukherjee, 1994;Nekesa et al., 2005).Also, soil pH affects the availability of nutrients to the plant which are needed for strong cell walls and resistance to fungal infestations.For instance, high levels of available calcium in more alkaline soils have been implicated in the resistance to root diseases caused by Pythium species (Paulitz, 2002).Ribosomal DNA sequences of the ITS region identified eleven Pythium species; P. aphanidermatum and P. splendens which were the most widely distributed species in the study locations.Other species confirmed include P. dissotocum, P. ultimum, P. irregurale, P. camurandrum, P. attrantheridium, P. graminicola, P. paroecandrum, P. acanthophoron and P. oligandrum.Previous studies conducted by Mukalazi (2004) and Nzungize et al. (2011) in Uganda and Rwanda, respectively identified some similar species that cause root rot disease.However, none of these identified eleven species have been previously studied and documented in Tanzania.Therefore, this study is the first to identify these Pythium spp. in common bean cultivation in Tanzania.
There was no association between the geographic distribution and identification of Pythium species within the collection area.For instance, P. aphanidermatum was found in altitudes of 1213, 1526 and 1754 m above sea level which is similar to the study conducted by Nzungize et al. (2011) that P. vexans was found in the highest number of districts where common beans were grown and this species was identified in low, intermediates and high altitudes of 900-1400, 1400-1650 and 1650-2300 m, respectively.In previous findings, similar studies identified P. apahanidermatum as a causal agent of root rot and crown necrosis of mature bean plants in Oman.This species was identified as the most aggressive and pathogenic species in the genus; it also has a wide host range that causes many economically important root rot disease (Al-Mahmooli et al., 2015;Ben Yephet and Nelson, 1999;Haritha et al., 2010).The most common species of Pythium that cause plant diseases of economic importance in Florida are Pythium myritylum and P. aphanidermatum and other species of Pythium that are sometimes associated with dysfunctional plants were P. splendens and P. irregulare.Also, P. splendens was identified from Eucalyptus grandis in northern Natal in South Africa and from soybean and corn in Ohio, USA (Dorrance et al., 2004;Kucharek, 2000;Linde et al., 1994).In eastern Washington, several species including P. attrantheridium, P. irregurale and P. paroacandrum were identified by using a real time PCR using collected soil samples (Li et al., 2014;Schroeder et al., 2006).This study also identified P. oligandrum from farmers' field within mono cropping system of common beans (Gichuru, 2008).
The phylogenetic analysis indicated diversity and similarities obtained from the alignment analysis of the ITS sequenced data using species-specific primers of 5.8S rDNA sequences.Specific proportional studies of the nucleotide sequences of rDNA genes provide a significant way of analyzing phylogenetic relationships over a wide range of taxonomic specie levels in fungal and non-fungal groups (Berbee et al., 1995;Harlton et al., 1995).
The spread of Pythium spp.occurs mostly through the movement of infested soil and plant materials by irrigation water, wind, farm equipment or animals.Heavy use of nitrogenous fertilizers and removal from farm products like plant residues after harvesting take alkaline nutrients off, tend to accelerate the rate of soil acidification and make favorable conditions for Pythium development.Therefore, farmers are advised to leave plant residues in the field after harvesting and use agricultural liming materials so as to increase soil pH from acidity to neutrality.This study provided information on the occurrence and distribution of Pythium root rot disease in two districts of Tanzania where common beans are grown.Xi K, Stephens JH, Hwang SF (1995).Dynamics of pea seed infection by Pythium ultimum and Rhizoctonia solani.Effects of inoculum density and temperature on seed rot and pre-emergence dampingoff.Canadian.J. Plant Pathol.17:19-24.

Table 1 .
Relative percentages of common bean cultivars grown in Lushoto and Mbozi districts.

Table 2 .
Categories of root rot symptoms in farmers' field.

Table 3 .
Period of occurrence of symptoms in farmers' field.

Table 4 .
Pythium species by geographical location and soil pH.