Morphological Characterization of Ugandan Isolates of Sphaceloma sp. Causing Cowpea Scab Disease

Scab is an important fungal disease of cowpea, affecting both young and old tissues including stems, leaves and pods of susceptible cowpea genotypes, leading to significant yield losses of up to 100% under severe infections. Colony characteristics on agar media, symptomatology, phylogenetic affinity of hosts and host range have been used to justify taxonomic distinctions. The correct identification and description of a pathogen is paramount in understanding its control or developing genotypes resistant to it. This study involved the isolation and culture of the scab fungus (Sphaceloma sp.) from infected plant parts (leaves and pods) collected from farmers’ fields across major cowpea growing districts and agro-ecological zones in Uganda. The fungus was characterized using growth habit on potato dextrose agar (PDA) media, conidia features, variability in radial growth rate (mm/day) among the isolates and pathogenicity and virulence of some isolates on 20 selected cowpea genotypes with different levels of resistance. A total of 495 Sphaceloma sp. isolates comprising of 419 from infected leaves and 76 from infected pods were obtained following isolation and culture. There was a wide variation in the isolates based on the amount, nature, colour, depth and rate of mycelia growth, features of conidia and number of septations. Based on the mean incidence, severity, AUDPC and pathogenicity on the 20 genotypes, the isolates were put into three pathogenicity groups. Isolates were mostly slow growing (> 14 days to cover entire 90 mm petri dish). Genotypes NE 31 and NE 70 showed broad spectrum of resistance to the isolates and could therefore be recommended as parental lines in the cowpea breeding programme to develop cultivars with wide horizontal resistance to the scab disease.


Introduction
Cowpea scab, caused by the fungus Sphaceloma sp., is one of the major diseases affecting the production of cowpea in Uganda. The disease is capable of causing up to 100% yield loss (Mbong et al., 2010;2012). Scab disease is widespread in all the cowpea growing districts of Uganda with mean incidence ranging between 35-70% and mean severity 2-4 out of a severity scale of 1-5, where 1 = no symptoms, and 5 = more than 50% infection, based on which Amuria and Tororo districts were reported to be hot spots of the disease in the country and was found to be more severe at higher altitudes (> 1200 m.a.s.l.) (Afutu et al., 2016a). plant pathology and many other disciplines (Barnett & Hunter, 1987). The genus Sphaceloma has been described by Barnett and Hunter (1987) as having disc-shaped or cushion-shaped acervuli, waxy; conidiophores simple, closely grouped or compacted, arising from a stroma-like base, sometimes almost appearing as a sporodochium; conidia hyaline, 1-celled, ovoid or oblong; parasitic; imperfect states of Elsinoe; similar to Gloeosporium and Colletotrichum. On the other hand, Ayodele and Kumar (2014) described the genus as having hyaline mycelium, scanty and submerged; hyaline to pale coloured conidia which are produced in pycnidia; have hyaline ascospores borne on the asci; pale coloured oblong to elliptical, and 3 septate. The conidia of isolates of the genus Sphaceloma were described as being small, unicellular, and hyaline, formed in more or less acervulus-like structures or, more commonly on continuous fertile layers of densely packed phialidic conidiophores with some species forming a larger, 0-2 septate, pigmented, thick-walled, spindle-shaped spores under certain conditions (Zeigler & Lozano, 1983). The conidia of the genus were later described as small, thin-walled, ellipsoid to (rarely) globose, commonly with one or two guttules and conidiophores being phialides hyaline to slightly pigmented 0-1 septate while conidiophores from the weedy species were phialides, hyaline brown 0-2 septate producing hyaline conidia (Alvarez et al., 2003).
The fungus attacks all the above ground parts of the cowpea plant (Emechebe, 1980;Iceduna, 1993;Mbong et al., 2010Mbong et al., , 2012. Infected leaves show spots on both leaf surfaces, cupped, appearance of small grayish lesions along the veins leading to leaf distortions and ragged appearance under severe infections while infected stems show oval to elongated silver grey lesions surrounded by red or brown elliptical rings with lesions coalescing and forming distortions under severe infections. On the other hand, sunken spots with grey centres surrounded by brown borders appear on infected pods, leading to malformation and formation of dark coloured pycnidia in the brown spots, with heavily scabbed young pods aborting or remaining attached to the plant as mummified black masses (Singh et al., 1997).
Screening of local germplasm for sources of resistance to the scab disease in Uganda showed a wide variability in response to the disease where lines which were rated resistant in one location, were found to be either moderately resistant or susceptible in another location (Afutu et al., 2016b). The variability in response to the disease, among other factors, suggested a variability in pathotypes of the scab fungus. This phenomenon complicates crop protection programs (Alvarez et al., 2003) and the development of resistant cultivars. This study was therefore, conducted to determine the morphological variability, growth rate and pathogenicity of some selected isolates on some local germplasm having varying levels of resistance to the scab disease to identify cowpea lines with broad spectrum of resistance for possible parents in the cowpea breeding programme.

Sampling, Isolation, Culture and Morphological Characterization of Pathogen
Samples of infected plant materials were collected in a field survey conducted in some of the major cowpea growing areas in the country, for isolation and culture of the scab fungus. Infected leaves and pods were cut into small portions using sterile surgical blades and were disinfested with 75% ethanol for 1 minute and then in 1% sodium hypochlorite for 1 minute, followed by rinsing in sterile water (Hou et al., 2014). The intact lesions were plated on commercial preparations of Potato Dextrose Agar (PDA) (Difco, Detroit) amended with Streptomycin Sulphate (1.5 g/L) and rose bengal (0.0025 g/L of agar) for isolation of Sphaceloma sp. (Mungo et al., 1998). Five lesions from each cowpea plant part were plated on the media in 9 cm-diameter Petri dishes and incubated at 26 o C for 5-7 days.
Following isolation and culture, a total of 495 pure fungal isolates from single conidium cultures were morphologically characterized based on colony characters such as the texture, density, colour, presence of conidial masses and colour of the reverse side of the dish (Talhinhas et al., 2002). The isolates were obtained from a total of 14 districts with each having three Sub-Counties and three farms per Sub-County making 126 farms in total. Isolates were named by assigning unique two-letter codes to represent the districts of origin and followed by serial numbers to identify the particular isolates (Table 1) and a letter "L" or "P" to indicate that the isolate was obtained from an infected leaf or pod respectively.

Pathogenicity of Selected Isolates
Five isolates were selected for pathogenicity tests based on the Agro-ecological zone and Districts where the crop was mostly cultivated. The number of isolates chosen from a region was based on the size of the region covered during the field survey and the morphological groups to which the isolate belonged. Pathogenicity of the five selected isolates was determined by inoculating seedlings of 20 cowpea genotypes (Table 2) selected on the basis of their resistance ratings following field screening under natural infections (Afutu et al., 2016b). Seeds of each of the 20 lines were sown in 20 cm diameter by 20 cm high plastic buckets filled with sterilized top soil  Vol. 8, No. 9;2016 Inoculum was prepared by culturing isolates in half strength (18 g/L) Potato Dextrose Broth (PDB) (HiMedia Laboratories Pvt., India) supplemented with Rifampicin antibiotic at 0.03 g/L, Streptomycin Sulphate at 1.5g/L and Rose Bengal at 0.0025 g/L. Glass wares containing the media and inoculum were put on a magnetic shaker (Stuart Scientific Flask Shaker SF1) set to 100 oscillations per minute (osc/min) for 10 days to prevent mycelia formation within the culture and to promote conidia formation (Waller, 2002). The concentration of inoculum was determined using Neubauer improved bright-light counting chamber (Superior Marienfell -Germany) and the concentration of inoculum was adjusted to 10 6 conidia/ml. Inoculation of plants were done 4 weeks after sowing by spraying leaves till run-off followed by covering inoculated plants with transparent plastic bags for 18 hours (Mchau et al., 1998). In the control treatment, sterile water was sprayed on the plants in place of the conidium suspension. There were four plants per treatment replicated three times and the buckets were arranged in a Completely Randomized Design (CRD).
Assessment of disease was done at 7 and 14 days after inoculation. A plant was considered positive for infection if a clearly distinguishable scab lesion developed, negative where no infections were developed while infections which were spotting but with no clearly identifiable scab lesions were designated as plus or minus (±) (Hyun et al., 2009). Disease severity was measured using a scale of 0-3 where 0 = no visible symptoms; 1 = very small pinprick type; 2 = small dark brown lesions with no chlorosis; and 3 = pale brown lesions surrounded by a chlorotic halo and with some distortion of the lamina (Mchau et al., 1998). Where there were any doubts about the identity of the symptoms, re-isolation was made from symptomatic plants inoculated with isolates. Note. a resistance rating by Afutu et al. (2016b): R = Resistant, MR = Moderately Resistant, S = Susceptible; b average data from two locations in 2014 season A (April-July); c average yield obtained from two locations (Afutu et al., 2016b).
Percentage pathogenic reactions of genotypes to isolates were calculated by expressing the number of isolates that caused clear scab disease symptoms (with the + symbol) over the five isolates (excluding the control treatment) and multiplied by 100 while percentage pathogenicity of isolates were estimated by expressing the number of genotypes with clear scab disease symptoms (with the + symbol) over the 20 genotypes and multiplied by 100. To estimate pathogenicity values for each isolate, pathogenicity codes (0-2) were assigned to the three symbols used in designating the presence or absence of scab disease symptoms (Hyun et al., 2009), where, 0 = "-" (no infections developed), 1 = "±" (for infections which were spotting but no clearly identifiable jas.ccsenet. scab lesion disease inc using arc Kurtosis-S using Mic (AUDPC) Campbell Where "t" "n" is the n mean seve al., 2011) Significan

Growth
The

Morphological Characterization of Pathogen
There was a wide variation in the isolates. Morphological characterization of the 495 isolates based on the amount, nature, colour, depth and rate of mycelia growth, nature or shape of conidia, number of septations and the colour of the base of petri dishes when inverted yielded six (6) morphological groupings (A-F) ( Table 3). Each of these groups had morphological structures similar to those described by Barnett and Hunter (1987) and (Ayodele & Kumar, 2014) but with slight variations in the parameters, hence, the different morphological groupings.   The amount of mycelia produced by the isolates ranged from very scanty (Group A = 14.1%) through scanty (Groups B, C and D = 37.8%) to abundant (Groups E and F = 48.1%) while the colour of colonies produced varied widely from white ( Figure 2F), pale (Figure 2A), white and peach ( Figure 2E), through to pink with red-brown to brownish-black pigmentations ( Figures 2B, 2C and 2D) confirming earlier reports that colony colour on PDA was found to be extremely variable even within the same isolates being differently pigmented under the same growth conditions (Zeigler & Lozano, 1983). The variable colour of the colonies observed in this study, including the dark to black pigmentations and the consistently high pigmentation has also been reported in some of the closely related species such as Elsinoe fawcettii, E. australis and Sphaceloma fawcettii, the causal organisms of scab diseases of citrus (Timmer et al., 1996).  Note. a Resistance rating based on Afutu et al. (2016b); RL = Resistance level; R = resistant; MR = moderately resistant; S = susceptible; b Pathogenic reaction of genotypes = number of isolates that caused clearly distinguishable scab lesions on genotypes out of the five isolates × 100; c Percent Pathogenicity of isolates = number of genotypes with clearly distinguishable scab lesions caused by each isolate out of the 20 genotypes × 100; d Pathogenicity value = summation of pathogenicity codes 0-2 based on the symptomatic effects of each isolate; e Pathogenicity groups: Isolates were separated into three groups based on the mean disease incidence, severity, AUDPC and the pathogenicity of isolates on the 20 inoculated cowpea genotypes, using Ward's cluster analysis (Alvarez et al., 2003) with 94% level of confidence.

Pathogenicity of Selected Isolates
The results of pathogenicity test of five selected Sphaceloma sp. isolates conducted on the 20 cowpea genotypes are presented in Table 4. The inoculation procedure proved satisfactory because susceptible genotypes were found to be consistently infected by the isolates though to varying degrees. Characteristic scab disease symptoms were observed on leaves of most infected genotypes by the time of the first observation (7 days after inoculation).
No scab disease symptoms were observed on stems even 14 days after inoculation of genotypes. Symptoms The use of radial growth rate has been shown to be a good measurement approach to differentiate isolates of the same species tested on similar types of media and the approach makes it easier to compare different data obtained within the same experiment or with other experiments to differentiate one isolate from the other or determine which media is best for growth of isolates (Miyashira et al., 2010). Note. Figures in parenthesis are error values; MG = Morphological group; AEZ = Agro-ecological Zone; *** = significant at P < 0.001. a, b and c represents radial growth rates measured at 7, 10 and 14 days respectively after inoculation of media (PDA) with 5 mm diameter disc plugs of isolates. d = days to full coverage of entire surface of the 90 mm petri dishes.
There was an association between radial growth rate (mm/day) of Sphaceloma sp. isolates and the District and agro-ecological zones from which isolates were obtained. The results of chi-square test of independence to test the hypotheses that radial growth rate of Sphaceloma sp. isolates were independent of Districts of origin and agro-ecological zones yielded the following test statistic values (χ 2 = 67.94, df = 26, and P < 0.001) and (χ 2 = 21.08, df = 4, and P < 0.001) for Districts and agro-ecological zones respectively (Table 8) hence the rejection of the two null hypotheses that growth rate of Sphaceloma sp. isolates was independent of districts and agro-ecological zones of origin. Out of the chi-square statistic of 67.94 for districts, isolates from Apac district contributed a margin of 14.869, out of which most (8.680) was due to fast growth rate which means that most of the isolates from the Apac district were fast growing, and covered the entire petri dish by the 8 th day. Isolates from two districts, viz., Kitgum and Yumbe showed similar growth rates, albeit from different ecological zones, as indicated by their contributions (9.839) each to the chi-square statistic. Also, out of the chi-statistic value of 67.94, slow growth rate contributed most (28.419) indicating that most of the Sphaceloma sp. isolates were slow growing (took > 14 days to cover the entire petri dish). Note. a Slow, moderate and fast growth rates implies isolates took 1-8, 9-14 and > 14 days respectively to grow to cover entire surface of petri dish (90 mm diameter); b AEZ = Agro-ecological zone, EAEZ = Eastern Agro-ecological zone, NESG = North Eastern Savannah Grassland, NWSG = North Western Savannah Grassland.
On the other hand, out of the test statistic value of 21.08 for the agro-ecological zones, isolates from the Eastern Agro-ecological Zone (EAEZ) contributed the most margin (12.478) out of which a greater part of it was due to moderate growth rate implying that most of the isolates from the EAEZ had moderate growth rate, thus, took between 9-14 days to cover the entire (90 mm) petri dish. On the whole, the observation that most of the Sphaceloma sp. isolates were slow (> 14 days) in growth rates (28.419 margin out of χ 2 = 67.943) as shown in the chi-square test between growth rate and districts of origin, was confirmed by the chi-square test of growth rate being independent of the agro-ecological zones of origin.
Thus, most of the margin (8.628 out of χ 2 = 21.08) was contributed by slow growth rate. This finding that the isolates were mostly slow growing is entirely consistent with earlier reports of studies on the genus Sphaceloma and its related genus Elsinoe (Zeigler & Lozano, 1983;Timmer et al., 1996).

Conclusion
The study revealed a wide morphological variation in the scab fungus (Sphaceloma sp.) occurring in Uganda with the isolates being grouped into six morphological and three pathogenicity groups. Growth rate of the fungus was found to be dependent on the District and Agro-ecological zones of origin and mostly slow growing. NE 31 and NE 70 cowpea genotypes showed broader spectrum of resistance to the isolates and could therefore be used to introgress resistance in a breeding programme to develop cultivars with wide horizontal resistance to the scab disease.