Screening of Selected Sorghum Genotypes for Resistance to Covered Kernel Smut Disease in Western Kenya

Sorghum is an important food security crop for arid and semi-arid tropics but its production is hampered by many biotic and abiotic factors including covered kernel smut disease (CKSD) caused by fungus Sporosorium sorghi in the Ustilaginaceae family. The disease attacks susceptible sorghum genotypes causing yield losses estimated at 43% in Western Kenya. This study determined the response of selected sorghum genotypes to CKSD under field and greenhouse conditions. A total of 15 elite sorghum genotypes were screened under field conditions in Migori and Homa Bay sites and under greenhouse at the University of Eldoret. Data on disease incidence and severity were collected per genotype and analyzed using R-Studio software and means were separated at 1% using Tukey’s test. Results showed significant differences among genotypes for disease incidence and severity under fields and greenhouse conditions. Disease incidence varied significantly (p < 0.001) among the genotypes ranging from zero (for T53, T30, IS3092, N4 and N68) to 64% (for Nyadundo2) under field conditions but ranged from 0-69% under greenhouse conditions. Similarly, severity followed the same trend with C26 having the worst attack with a score of 5 while T53 recorded the least (score of 1). This study has identified potential sources of resistance for covered kernel smut disease that can be utilized to manage the disease and significantly improve sorghum yields in the target regions.


Introduction
Sorghum (Sorghum bicolor (L.) Moench) is ranked fifth in importance among cereals in the world and is a major food crop for developing countries (FAO, 2012). It is particularly important in areas with high temperatures and low rainfall due to its resilience (Hayden, 2002). The sorghum grains can be used for syrup production, making of leavened and unleavened bread, bio-energy, bio-ethanol production and preparation of alcoholic beverages (Tonapi et al., 2020). In Sub-Saharan Africa, Middle East, North Africa and India sorghum is mainly used as human food while in Europe, Australia, China, and Western Hemisphere countries it is used as animal feed, forage, and for industrial purposes including ethanol production. Sorghum production is mainly concentrated in Asia, Sub-Saharan Africa and the Americas and Caribbean (FAO, 2012).
Its global consumption is estimated to be 61.0 million metric tons per year (USDA, 2019). However, grain yield in most parts of the world is relatively low, estimated at 0.925 t/ha compared to 5 t/ha reported from experimental stations (ICRISAT, 2004). The low yield is attributed to a number of factors including biotic, abiotic and socio-economic factors (Esele, 2013). The most important diseases and pests of sorghum include shoot fly, stem borer, shoot bug, aphids, sorghum midge, head bug and covered kernel smut disease. Collectively, these constraints limit sorghum production and hamper its productivity across regions of the world (Tonapi et al., 2020).
Covered kernel smut disease (CKSD) caused by Sporisorium sorghi in the Ustilaginaceae family is a major constraint in sorghum production (Mtisi & McLaren, 2008). The fungus is seed-borne and develops systemically as the sorghum crop grows. In Kenya, its incidence is exacerbated by the informal sorghum seeds system whereby small-scale farmers continue to share and exchange retained own untreated seeds for planting the next season's crop among the communities (Gwary et al., 2007). According to Howard et al. (2005) maturing fruiting bodies of the fungus called sori rupture and release teliospores that infects seeds on the same or other sorghum plants. The teliospores of the fungus replace the grain in the panicle causing direct crop losses in grains.
According to Sisay et al. (2012) the fungus Sporisorium sorghi in the Ustilaginaceae family can grow and develop at 10-32 ºC, but the optimum soil temperature conducive for the disease development is 18-25 ºC. The infection is established in warmer and wet soils with humidity of between 15-20%. More importantly, periods of delayed seed germination and emergence are optimal for the infection (Ashok et al., 2011) which enhances its incidence. In the year 2012, Gautam et al., recorded more than 50% disease incidence in Ethiopia. In general disease incidence varies from place to place. Annual yield losses due to CKSD in Africa reaches 10% with localized losses of 60% or more (Sisay et al., 2012). In Kenya, CKSD also causes significant yield losses ranging from 42-48% (Okongo et al., 2019). However, in Migori and Homa Bay Counties, little is known about its incidence, severity and distribution. Some new improved sorghum varieties that were introduced in the area by the Rongo University Sorghum Improvement Team in 2017 were infected by the disease (http://www.ccrp.org) raising the issue of its management.
To minimize yield losses due to CKSD, several methods can be used such as chemicals, cultural, biological and through breeding for tolerant crop varieties. Chemical method includes the use of fungicides such as Captan and Carboxin+Thiram (Vitavax) which assist in reducing the incidence and severity of the disease on sorghum but does not completely control the disease (Jere, 2004). Moreover, most of these fungicides are extremely expensive and unaffordable to the smallholder farmers.
Several cultural methods are available for controlling the disease including soaking of seeds in water for four hours followed by drying of seeds under shade, collection of smutted ear heads and incinerating them (IPM, 2008). According to Adane and Gatam (2000), CKSD can also be controlled by use of fermented cattle urine and botanicals from Abeyi (orm) Maesa lanceolata. However, the two methods are not widely used and their efficiency in different regions need to be established. Moreover, Abeyi plant is not readily available for farmers in Western Kenya (Okongo et al., 2019).Therefore the CKSD remains a major threat to food security in western region despite the chemical, biological and cultural methods currently in use owing to their labour intensive nature and or cost.
The use of resistant genotypes is one of the most viable strategies for the control of covered kernel smut disease (Kutama et al., 2013). This is because orphan crops like sorghum has a low return to investment and therefore, the introduction of resistant varieties remains the most cost-effective and sustainable option to control covered kernel smut disease (Wilson, 2011). In Kenya, there is lack of smut resistant genotypes, creating a need to identify stable sources of resistance through screening which could be utilized directly or used in breeding programs to develop other resistant varieties. Therefore, this study seeks to improve production of sorghum through screening and selecting resistant genotypes to be used for management of covered kernel smut disease.

Plant Materials
A total of 15 plant materials were used which included the newly released sorghum varieties by Rongo University Sorghum Breeding Program, commercial variety and farmers' cultivars (Table 1)

Description of Experimental Sites
The study was conducted in two counties, Homa Bay and Migori. The first site is located at 0 o 42′S and 34 o 50′E, 1221 m above sea level, has an average annual temperature of 21.2 o C with a humidity of between 20-28% and annual precipitation of 1369 mm per year with Vertisol soil type. Migori site is located at 1 o 07′S and 34 o 42′E. It has an elevation of 1281 m above sea level with daily temperature ranging between 26-34 o C with humidity of between 18-20% and average annual rainfall estimated at 1100 mm with granite type of soil.
The greenhouse screening was done at the University of Eldoret located at 0.52 o N and 35.27 o E which has an elevation of 2090 m above sea level, average temperature of 15.8 o C and average rainfall of 1263 mm.

Preparation of Sporidial Inoculum Suspension
The CKSD inoculum was prepared by collecting 5 grams of dry teliospores from mature panicle smut infected sorghum genotypes from on-farm trials by shaking them out of the heads and sieving to remove the debris, The teliospores were then washed in 70% ethanol to sterilize then suspended on 250 ml sterile water and plated on Potato Dextrose Agar (PDA) and incubated in the dark at 28 o C for 3 days. The sporidial colonies were then transferred in flask containing 100 ml potato Dextrose Broth (PDB) and incubated on a rotary shaker for 4 days. The suspension was then filtered using a cheese cloth, which was then used to inoculate the seedlings with a hypodermic syringe according to procedures described by Frederiksen (2000).

Germination of Sorghum Seeds in Pots
Ten seeds of each of the fifteen sorghum genotypes (Table 1) were planted and grown in pots arranged in a Completely Randomized Design (CRD) replicated three times in the greenhouse, each pot was filled with 1.5 kg forest soil + 0.15 g teliospores and mixed with a handful of organic matter. The seedlings were then thinned when they were I month old to three seedlings per pot.

Inoculation of Seedlings
The inoculum suspension was then used to inoculate the seedlings with the help of a hypodermic syringe when they were 10 cm in height (4 weeks old). An inoculum suspension was injected into each seedling continuously until drops of the inoculum were seen at the top of the leaf.
For field screening, fifteen genotypes (described in Table 1) were planted in CKSD hotspots in Migori and Homa Bay sites. The experiments were set up in a Randomized Complete Block Design (RCBD) with three replications. Each genotype was planted in a (2.25 × 4) m plot with 4 rows at a spacing of (75 × 20) cm. Standard agronomic practices were followed to raise a healthy crop.

Covered Kernel Smut Disease Incidence
This was assessed on infected panicles by determining the proportion of sorghum plants showing the symptoms of the covered kernel smut disease compared to the total number of plants in the plot, and the incidence expressed as a percentage as described by Chaube and Punder (2005)   Data collected on disease severity and incidence was transformed using square root transformation method and analyzed using R-Studio. Analysis of variance was done for the two sites and the greenhouse according to K. Gomez and A. Gomez (1984). Differences were accepted as significant at p < 0.001 and the means separated at 1% using Tukey`s range test.

Disease Incidence Under Field Conditions
At Migori site, there were significant differences(p < 0.001) on the incidence of covered kernel smut disease amongst the fifteen sorghum genotypes (Figure 1, Table 3) but replication and residuals had no effect on disease incidence, Four varieties namely N4, MUK24, N68 and IS3092 showed significant variation in disease incidence compared to the rest of the varieties. C26 had the highest mean disease incidence (60%) compared to N68 (3%) while the local checks (Ochuti and Jowi), Nyadundo1 and Nyadundo2 showed statistically similar disease incidence.
At Homa Bay site, there were significant differences (p < 0.001) on incidence of covered kernel smut disease amongst the fifteen sorghum genotypes (Figure 2, Table 3). Replication and residuals had no effect on disease incidence levels. Ochuti and Jowi, the local checks had the highest mean incidence of 56.7% while IS3092 had the lowest mean incidence of 3% (Figure 1b). Nyadundo2 and Nyadundo1 had a mean incidence of < 50% which compared well with the commercial checks, Seredo which showed a mean incidence of 43.3%.

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Conclusion
The study has identified sorghum genotypes that are tolerant, moderately tolerant and susceptible to covered kernel smut disease in Western Kenya through field and greenhouse screening. All the commercial and farmer varieties were found susceptible to the CKSD. The tolerant varieties included MUK27, T53B, N68, T30B, E117B, MUK157, IS3092 and N4 while the susceptible ones were, Nyadundo 1 and 2, Ochuti, Jowi and C26.The observed large variation in incidence and severity indicates possibility of managing the disease through selection and breeding for resistant varieties. We recommend further breeding for genetic improvement of sorghum using the identified resistant lines.