Seed Health, Quality Test, and Control of Seed-borne Fungi of Some Improved and Local Cultivars of Rice (Oryza sativa L.) in Kano, Northwestern Nigeria

This research was carried out to evaluate the health and quality of rice seed. The germination of seed and presence of rice seed related fungi were recorded and used to evaluate the eff ect of seed dressing chemicals on germination and vigor index over untreated rice seeds. Seven cultivars commonly grown in Kano, Kano State, Northwestern Nigera “FARO 52” (WITA), “FARO 44” (SIPI), “FARO 60” , (improved varieties), “Kwandala” , “Jamila” , Ex-china, and “JIF” (local varieties) were used in this study. The seed dressing chemicals used were Apron Star 42 WS, Dress Force 42WS and ZEB-Care 80%WP. This study was performed under three main tests, i.e dry inspection, blotter tests, agar plate and microscopic examination. The highest number of healthy seeds (94.16%) was recorded from “JIF” variety and lowest (64.77%) from “Jamila” . The highest number of deformed seeds was observed from variety “FARO 44” whereas the lowest noted on “JIF” . The identifi ed fungi were Fusarium spp., Bipolaris oryzae, Aspergillus fl avus, Curvularia lunata, Aspergillus niger, and Nigrospora oryzae., Rhizoctonia spp. and Rhizopus spp. Highest seed infection was recorded for A. fl avus, A. niger, and Fusarium spp., and the least with C. lunata and N. oryzae. Treated seeds with Zeb-care (Mancozeb 80% WP), increased their vigor index over untreated by 62.78% and can be recommended as seed dressing chemical for optimun control of rice seedborne pathogens.


Introduction
Rice is considered as one of the major staple cereal foods and forms a signifi cant component of the energy resource of the human race. Rice is an economically and scientifi cally important crop and mostly produced and consumed in Asia and African countries (Islam et al. 2000;Dede et al. 2019). Currently, 35-40 % more yield productivity is required to meet global demands (FAO 2017). The available rice stock in these countries is stored to adjust the demands and supply for both seed and food (Islam et al., 2000). To date, the seed of other cereals crops like wheat, maize, and barely have received adequate attention for storage and post-harvest, however, the research for rice is still not completely yet (Misra et al., 1995).
Storage facilities in Asia and Africa are still far from satisfactory. The farmers keep their stock in various ways, some of the means of storage are out-ofdate; therefore, weak and practically unserviceable. Increasing rice production and subsequent reduction of its international importation and endemic plant pathogens continue to be a challenge in safeguarding plant health in Nigeria. Therefore, early and accurate diagnosis and pathogen surveillance is the most important approach to tackle this matter. The assessment of seed health standard in rice is very important for farmers and food security as it is a fi rst line approach in managing seed-borne diseases of plants.
Furthermore, the quality of planted seeds has a critical infl uence on the ability of crops to become established and to realize their full yield potential and value (McGee 1995). The importance of seed quality in understanding the full potential of a variety is well known. The three signifi cant aspects of seed quality are genetic and physical purity, high germination percentage and vigor, and free from seed-borne diseases and insects (Seshu and Dadlani 1989). Seed vigor is recognized as an essential seed quality parameter distinct from germination (Seshu et al., 1988). Seed-borne diseases may not only introduce new pathogens to aff ect the quantity or quality of the crop yield but also contaminate the soil permanently (Anselme, 1981).
Fungi are commonly identifi ed as causal agents of many seed-borne diseases (Nsemwa and Wolff hechel 1999). Studies from Biruma et al. (2003) and Kanobe et al. (2004) showed a wide range of seed-borne fungi pathogens on the farmer's stored rice seeds, of which many farmers mainly rely on to propagate the next planting session. Most seedborne diseases caused by the fungi pathogens are disastrous as they may decrease seed germination, causing seed discoloration and produce toxins that may be detrimental to man and domestic animals. Distribution of some seed-borne fungi associated with rice seeds has been reported in many countries, including Nigeria (Suleiman and Omafe 2013); Pakistan (Butt et al. 2011); Egypt (Madbouly et al. 2012); Bangladesh (Ora et al. 2011); Cameroon (Nguefack et al. 2007) and Chad Republic (Serferbe et al. 2016).
The main objectives of this study were i) to identify seed-borne fungi associated with rice and evaluate its role for seedling abnormalities ii) to investigate the possibility of combining germination and seed health tests to assess the infl uence of seed treatments on germination and seedling vigor.

Experimental Site
The experiment was carried out at the Research and Teaching laboratory and screen house of the Department of Crop Protection, Faculty of Agriculture, Bayero University, Kano, (11° 58 " N, 8° 25 " E and 47m above sea level) Northwestern , Nigeria.

Detection of Seed-borne Fungi using Blotter Method
To perceive the seed-borne fungal pathogens associated with the seeds using blotter method was used following the international rules for seed testing as used by Ahmed et al. (2013). modifi ed from ISTA to perceive the seed-borne fungal pathogens associated with the seeds. In this method, three layers of blotting papers (Whatman TM fi lter paper No.1) were soaked in sterile distilled water and placed at the bottom of the 90 mm diameter plastic Petri dishes. Four hundred seeds from each sample were taken randomly and then put on the moist fi lter paper at the rate of 25 seeds per plate. Incubation was conducted at 28 ± 2°C under 12/12 alternating cycles of light and darkness in the incubation room for seven days. During the incubation period, Petri dishes were checked and wetting of fi lter paper done as the need arise. After incubation, seeds were examined under the stereomicroscope for the presence of seed-borne fungi. Number of infected seeds expressed in percentage. Two selected rice varieties within the highest disease incidence were tested on agar plate method and seed treatment.

Identifi cation of Fungi
Each seed was observed under stereo-microscope at ×16 and × 25 magnifi cations to identify the seedborne fungi. The most associated fungi were detected by observing their growth characters on the incubated seeds and identifi ed following the identifi cation keys from (Ahmed et al., 2013). Temporary slides were also prepared and observed under a compound microscope for proper identifi cation. The fungi were identifi ed up to species level, following the identifi cation keys of . The results presented as percent incidence for the individual fungal pathogen indentifi ed.

Agar Plate Method
In this technique, one hundred seeds tested for each variety and replicated ten times. Surface sterilized seeds as above were plated (10 seeds/Petri dishe) on the potato dextrose agar (PDA) medium. The plated seeds were incubated for seven days at 28 ± 2 °C in darkness. At the end of the incubation period, fungi developed from the seeds on the agar medium, and then were sub-cultured into fresh medium for sporulation. The fungi were examined and identifi ed based on colony characteristics, and morphology of fruiting bodies under a compound microscope after seven days incubation.

Examination of Incubated Materials
The slide of various fruiting structures of the fungi was prepared and observed under a compound microscope (× 100) for identifi cation following (Barnett and Hunter, 1998).

Fungicide Seed Treatment
Seeds were rinsed with sterile distilled water in a clean plastic bowl to remove impurities before chemical seed treatment. Fungicides were applied to the seeds following the manufacturer's recommendation. The three fungicides used are Apron Star 42 WS (20% w/w thiamethoxam + 20% w/w metalaxyl-M + 2% w/w difenoconazole), Dress Force 42WS (20% Imidacloprid + 20% metalaxyl-M + 2% tebuconazole) and ZEB-Care 80%WP (mancozeb 80%WP). 0.025g of each chemical used in treating 10g of rice seeds of "Jamila" and "FARO 44" varieties with a high percentage of disease incidence that had been selected for this experiment. The control consisted of untreated rice seeds, that were allowed to dry after treatment.

Germination of Treated-Seeds in Plastic Pots
Planting media used in the screen house was sterilized sandy soil so as to meet ISTA regulations for grain size, pH, and conductivity (only recommended for seeds >5mm). Plastic pot (30 cm diameter) 10 L in volume and about 16.5 cm depth was used. The treated seeds have been supplied with suffi cient water to prevent from drought condition.

Vigor Test
A vigor test was done using the sand pot method (ISTA 2001). Shoot and root length were measured using a ruler (30 cm length) after 14 days of sowing. Fifteen seedlings (30 seedlings per treatment) were randomly selected for the measurement of shoot and root length. The seedling vigor was determined using the formula of (Abdul-Baki and Anderson, 1972): Vigor index = (mean of root length + mean of shoot length) × % of seed germination.

Data Analysis
All the collected data was analyzed using descriptive statistics and presented as percentage (%).

Dry Inspection of Rice Seed Samples
The highest number of visually healthy seed (94.16 %) recorded from "JIF" variety and lowest (64.77 %) from "Jamila" , respectively ( Table 1). The highest number of deformed seed (1.8 %) recorded from variety "FARO 44" , and lowest (0.47 %) recorded for "JIF" . The highest number of apparently healthy seed and lowest deformed seeds were observed in the seeds of "JIF" variety (Table 1), suggesting it as the best among all the varieties.

Fungal Pathogens Identifi ed from Rice Seed Samples
The present study has also focused on the survey of seed-borne fungi associated with rice varieties mostly grown in Kano state, Nigeria. Some of the fungal species were isolated and identifi ed with the blotter method while others were obtained by the agar plate method. During our investigation, eight species of fungi identifi ed on the seeds of seven rice varieties ( Table 2). The identifi ed fungi were Fusarium spp., Bipolaris oryzae, Aspergillus fl avus, Curvularia lunata, Aspergillus niger, and Nigrospora oryzae., Rhizoctonia spp., and Rhizopus spp. Highest seed infection recorded for A. fl avus, A. niger, Fusarium spp., followed by Rhizoctonia spp and Rhizopus spp., while B. oryzae, C. lunata, N. oryzae recorded the lowest percentage of seed-borne infection. The Aspergillus species, the one recorded with high frequency, tends to produce toxic substances apart from the deteriorating eff ect of root rotting. This result was in consistance with fi ndings of (Javaid et al., 2002;Ibiam et al., 2006), that reported the presence of Aspergillus species but, not as main pathogen. Many of the isolated fungi from our study have been reported to be associated with seeds of other crops (Tsopmbeng and Fomengia, 2015). Some of them are also known to cause seed rot, decrease seed germination and cause pre and post damping off as well as seedling death (Al-Kassim and Monawar, 2000) when Fusarium spp and Aspergillus fl avus are found in high frequency. This high frequency of the detected seed-borne pathogen from our study has been reported in previous studies on the seeds of rice in India (Archana and Prakash 2013) and Pakistan (Khan et al., 1988;Javaid et al., 2002). Soil borne mycofl ora associated with rice and their infl uence on growth was also recorded in Abakaliki, Ebonyi State in Southeastern Nigeria (Utobo et al., 2011). Although, Note: HS = healthy seed = good looking seed free from spots and abnormalities, DS = discolored seed, SS = spotted seeds, BS= broken seeds, IDS = insect damaged seeds, CS = chaff y seeds, DES = deformed seeds, VM = varietal mixture and IM = inert matter no such kind of soil borne pathogens related to rice crops were recorded in our study area.

Quality Test: Purity Analysis of Rice Seed Samples
A seed purity analysis was conducted and concluded: 100 % pure seed in "FARO 44" rice variety, and the lowest pure seed (95.18%) was recorded in variety Ex-China (Table 3). The lower purity of cultivar Ex-China compared to other cultivars was caused by lousy storage practices by the farmers while high purity "FARO 44" was obtained from applied seed technology in commercial company. Another study from Chowdhury's (2012) about seed quality status reported that HYV has the highest percentage of purity. Haque et al. (2007) also found 99.01 % pure seed from the trained farmers and minimum (96.19 %) in untrained farmers. Percentage of seed purity was determined by Uddin (2005) in Upazilla under Noakhali district that ranged from 95.59 to 99.39 %. In convex with our fi ndings,  categorized the percentage of pure seeds ranging from 91.20 to 98.89 % collected from trained farmer's stored rice seed.

Germination Test Using Blotter Method of Rice Seed Samples
The result in Figure 1 shows the mean percentage of germination of diff erent rice seeds, ranged from 0 % to 90.50%. The highest germination was recorded in variety "JIF" (90.50%), while the lowest germination found in variety Ex-China (0%). The variety "Jamila" has the highest percentage incidence of fungal mycelia growth examined using blotter method (14.25%), and variety Ex-China recorded the lowest rate (1.25%). This research clearly showed that "JIF" has the highest germination percentage even though it has been sourced from farmer-stored seed within the study area. The zero germination in the variety Ex-China could be a result of poor storage conditions that lead to the development of seed-borne pathogens, insects, mechanical damage at both handling and storage, or aging which renders the seed to be not viable or dormant. Some of these fungal pathogens are known to cause seed rot, decreasing of seed germination, pre and post damping off , and seedling death (Al-Kassim and Monawar, 2000). According to Lamrani et al. (2013), Alternaria padwickii colonizes variety of seeds thus reducing the percentage germination and causes seed rot. Species of the genus Curvularia, in particular, C. lunata has been reported to infect the embryo of the seeds, therefore, reducing the percentage germination of rice seeds (Imolehin, 1987;Bautista and Opina, 1991). C. lunata was reported in diff erent rice varieties and identifi ed as one of seed-borne pathogens (Butt et al., 2011;Utobo et al. , 2011;Ashfaq et al., 2015).

Eff ect of Seed Treatment on Seed Vigor Index
The variety "FARO 44" and "Jamila" were selected as test sample of seed dressing trial using three diff erent chemicals due to the facts they are most commonly grown variety in Kano. "FARO 44" is an improved variety while "Jamila" is local. Another consideration is these both varieties have higher mycelia growth than others and this indicator is suitable to measure the increasing of vigor index based on the chemical treatment.
The vigor index of treated seeds using Zeb-care was increased up to 62.78% over the untreated control (Table 4). The control (untreated variety) from "Jamila" has a vigor index score of 928.71, while "FARO 44" has the vigor index of 904.11. Variety "Jamila" treated with Zeb-care has 991.49 vigor index and it indicated that there was an increase of vigor index with 62.78% followed by Zeb-care treatment on "FARO 44" with an increase of 31.78%. The lowest increase in vigor index (5.28%) was found in variety "FARO 44" treated with the Apron star (Table 4). The variety "FARO 44" treated with Dress force shown a negative value of -6.11 over the untreated which directly implies that there is a decrease in vigor index over control. Following the obtained values, the effi cacy of seed dressing chemicals was hypothesized benefi t the variety "Jamila" more. The Zeb-care dressing chemical displayed a more positive eff ect (+31.78 and +62.78) on both cultivars ("Jamila" and "FARO 44") followed by Apron star (+5.28 and +31.17) and lastly Dressed force (-6.11 and +22.40) respectively.
Similar result also has been reported on previous studied by (Bhuiyan et al., 2005;Hossain and Hossain 2012) whereas vigor index increased in vegetable treated seed using BAU Bio-fungicide. Furthermore, Shultana et al. (2009) also evaluated wheat seeds treated with Bavistin and showed higher vigor index on 2843 followed by BAU Bio-fungicide treated seed on 2661.

Conclusion
Based on the result obtained in this experiment, it can be concluded that farmers are strongly advised to use any of the following varieties "JIF" , "Jamila" and "FARO 44" in Kano State as it has been tested and found to be suitable in both seed quality and purity, it will also help to improve rice production and reduce the threats due to seed-borne fungal diseases. The use of Zeb-care treated seed, which is often neglected by farmers, is encouraged as the seed dressing fungicide (Zeb-care 80WP) signifi cantly increased seed germination and seedling vigor.