PROPOSAL TITLE: Evaluation of Rice (Oryza sativa L.) Seed Systems and Quality Improvement in Liberia

School of Agriculture and Biotechnology, Department of Seed, Crop and Horticultural Sciences

 Proposal Submitted to GTA Research Fellowship at RUFORUM Secretariat for Research Grant

 Ousman Sarlia Dorley

ADM. No: SAGR/SCH/P/003/19

2020

Executive Summary

Lack of adequate quality seeds and poor pre and post-harvest handling techniques continue hampering rice production in most developing nations, especially in Africa. The poor quality seed accounts for more than 50% rice grain losses. The decline in rice yield poses a severe food security issue in countries where the crop is considered a staple food. In Liberia, rice is dependent upon as staple food by more than four million people. A continuous decline in the country’s production has forced the government to import about 70% of the rice accounting for US$200 million annually national expenditure. Therefore, the unsustainability of such annual spending informs this study, which aims to; evaluate rice farmers’ awareness on pre- and post-harvest practices and identify fungal pathogens affecting rice seed quality under various agro-ecological zones, and evaluate the efficiency of some local botanicals in enhancing seed quality. The exploratory survey, comprising of a mixed structured questionnaire will be administered to purposefully sampled rice farmers in the six counties of Liberia; Bong, Gbaporlu, Montserrado, Nimba, Lofa and Grand Gedeh Counties to get insights on their seed quality awareness. Alongside questionnaires, sample rice seed will be collected for further research. Sampled seeds will be subjected to fungal screening to identify pathogens affecting the seeds in respective counties. After identification of the pathogens, the seeds will be stored in different botanical substances to evaluate the influence in hindering pathogen prevalence. The powders of botanical’s neem (Azadirachta indica) (roots, barks, leaves), ginger (Zingiber Officinalis) garlic (Allium sativum) and common bean ash composed with synthetic fungicide Benlate will be subjected to in-vitro testing to determine the best range of each substance in inhibiting Aspergillus. flavus and Penicillium citinum growth. Survey data will be subjected to descriptive analysis, chi-square correlations, and Phi and Cramer analysis to determine the level of awareness and seed quality assurance practice in each county. Sampled seeds will be treated with the botanical substances in a completely randomized design. Data obtained will be subjected to descriptive analysis combined with principle component analysis. Interaction of the combined effect of the botanical powders will also be determined by the use of multiple analysis of variance (MANOVA). The study results are intended to benefit the low-income farmers, improve rice production by enhancing seed quality, and inform the policymakers and stakeholders to optimize rice production in the country.

CHAPTER ONE

1.0 INTRODUCTION

1.1 Background information

Rice (Oryza sativa L.) is the most important cereal crop in terms of consumption and production globally, followed by corn and wheat (Cosslett & Cosslett, 2018). Globally, China is the leading rice producer with more than 200 million tons of rice grain in 2018 (FAOSTA, 2020), while Nigeria is the major rice producer in Africa followed by Egypt with 6.8 and 4.9 million tons of grain rice production in 2018 respectively (FAOSTAT, 2020). Production and consumption are projected to increase due to the burgeoning population, and climate change restrains the production of other cereals. Rice boasts of multi-sectoral utilization leading to diversified modes within its value chain. Besides human consumption, rice is also used as animal feed (fish, poultry, and pigs) and as the source of medicinal oil extracted from the outer layer of the grain, among others.

In Liberia, the crop is a primary preferred staple food for the population, (Hilson & Bockstael, 2012), dependent upon by more than 4,195,666 people (the Republic of Liberia, 2019). Liberia rice’s annual per capita consumption is estimated to be more than 140 kg per capita in 2010 (Chauhan, Jabran, & Mahajan, 2017), leading in Africa. The crop is mainly produced by smallholder farmers in many parts of the country (Ashmun, 2020). Rice cultivation in Liberia is less intensive. It is characterized by low inputs, which do not meet the essential crop requirement in terms of agronomic, technical, and post-harvest handling strategy for the crop. Hence, translating to an annual decline of the yield.  For instance, in 2018, grain production was 257,995 tons produced from 238090 hectares of land averaging to 1.083603 compared to Nigeria 2.035084 tones (FAOSTAT, 2020).

In the past decade since the end of civil war (Vorrath, 2018), the area under rice production has steadily increased. And many people have joined rice farming, making it the best sector employing more than 51% of the Liberian population (Knoema, 2019). In recent years concerted efforts between the government, world bank, and non-governmental organizations have seen the rice sector received support in terms of agriculture equipment and other inputs such as fertilizers as well as improved varieties (MOA, 2014). The efforts indicate acknowledgment of the stakeholders of the rice role in national food security. Rice production has continuously declined in comparison to the area under production despite the synergies created by value chain stakeholders and farmers’  increased involvement (MOA, 2014).

The optimal production is hindered by factors such as poor pest management, inappropriate agronomic practices, low fertilizer use, pre- and post-harvest losses, marketing challenges, and, most importantly, inadequate quality seeds (FAO, 2019a). It has been confirmed by research that seeds account for 50% of the crop yield. Thus, the seeds’ quality and viability is a crucial aspect of rice production (Neergaard, 1997). The rice seed quality in Liberia is affected by a lack of proper seed system and inappropriate handling techniques, which favors the prevalence of fungal attack (ISSD-Africa, 2017).

The common seed fungal contamination is caused by Aspergillus flavus and Penicillium spp (Amadi & Adeniyi, 2009). The two fungi species thrive in improperly stored seeds exposed to moisture conditions. Besides hugely decreasing the seed quality, the fungi pose health risks to humans (Reddy, Reddy, & Muralidharan, 2009). According to FAO (2019b), the two are the primary culprits producing aflatoxins in grains, which is a chemical responsible for many cancer cases in developing nations.  Therefore, there exists a need to develop economically feasible and practical strategies that will aid farmers in ensuring rice seed quality.

This work will, therefore, provide feasible and practical strategies that will aid farmers in ensuring rice seed quality.  Thus, the study will be conducted in the main rice-producing agro-ecological zones of Liberia to establish the seed quality assurance practices employed by farmers. Using a survey that will utilize a purposive combined open and closed-ended questionnaire will collect both farmers’ information and seed samples. Secondly, collected seed samples will be screened for fungal and bacterial infections. Finally, sustainable seed quality management practices will be investigated through subjecting the seeds to different levels of botanicals to evaluate their efficiency in maintaining seed quality under field conditions and a confirmatory test in the laboratory.

1.2 Statement of the Problem

Limited access to quality seed rice is a significant challenge for rice production in Liberia. Provided that the farmers involved in cultivation are poor and illiterate (MOA, 2014), thus, ensuring seed quality is a significant challenge that has been mirrored in a continuous decline in crop productivity (Kontgis et al., 2019). Stakeholders’ efforts to revive production and curb the increased imports of the commodity has seen little success due to unstainable seed system implicated by inadequate information on quality assurance practices employed by farmers in Liberia.

Since the end of the country’s civil war (2003), the government has strived to stabilize food production by targeting the high potential areas using the most preferred food crops. Crops such as cassava and plantain have recorded tremendous success compared to rice. This is because rice is a sensitive grain crop whose optimal production can only be achieved by optimizing agronomic practices alongside conducive climatic factors. Despite the country’s less depleted soils and the right amount of rainfall, the poor rice yield is embedded on the farmers’ use and reuse of poor quality seeds and stagnated awareness on economically feasible means of ensuring seed quality.

Climatic characteristics facilitate grain seed quality deterioration in tropical regions. Research indicates an annual loss of a third of the produced to post-harvest handling globally (Affognon et al., 2015). Liberia being the wettest country in Africa, is not spared of the spoilage, and according to Lidow (2011), the county losses about half of its total production to waste. While more than half of national rice production spoils through handling.

Fungal invasion of rice seeds, especially Aspergillus flavus, Rhizopus stolonifera, Fusarium culmoreum, and Penicillium spp are among the significant biotic factors constraining rice production in tropical regions especially Liberia, by pre-harvest effects on crop vigor and reduction of the quality of seed grains (Oyetunji et al., 2012). Management of such fungi has failed in Liberia due to a lack of information regarding the severity and prevention of the fungi, specifically under the Liberia climatic and environmental conditions (Yacouba Séré et al., 2013). Affected grains, when used as propagation materials easily, recycles the fungal into the subsequent crop generations, which impacts both productivity and human health negatively.

1.3 Justification of the study

Liberia’s human population has steadily grown since the end of the civil war in 2003 (Vorrath, 2018). While this scenario has been associated with a positive impact on the national labor, giving rise to skilled, semi-skilled, and unskilled cheap local labor, the challenge intertwined in these population growths is increasing in poverty. The young population is therefore forced entirely dependent on agriculture for both food and other basic needs. Rice, as a significant crop, has been key to the achievement of these goals. Unfortunately, its decline in yields despite an increase in cultivation area threatens the livelihood of almost all Liberians and, most notably, resource-poor farmers.

Figure 1. FAOSTAT. The trend of Rice yield and the land under cultivation of rice for the year 2008-2018

Rice forms Liberians staple food in terms of consumption and ranks first as the most consumed cereal estimated to provide more than 85% of calories in the country (MOA, 2014). The inability of the local production to sustainably supply the grain relative to its demand has made the country to direct most of its budget expenditures towards importation. Overreliance on imports accounts for more than two-thirds of consumed rice, translating to more than US$200 million annually (the Republic of Liberia, 2019). These figures are too high to be sustained by the Liberian population, as the Liberian community averagely lives on less than the US $1 a day (FAO, 2019a). Imports have made access to the commodity delicate due to their high prices hence threatening the country’s food security.

Such challenges thus form the basis of this study. Understanding the level of farmers’ ingenious knowledge on rice seed quality maintenance will highlight the weak links that both the government, policymakers, and other stakeholders need to address with urgency to shift the current production trajectory. Evaluation of local botanicals concerning the increased shelf life of the seeds will ensure that farmers access new information that will help them cheaply ensure they possess quality seeds. The study further aims at the identification of the common fungal pathogens affecting rise grains to provide information to steer targeted management practices.

1.4 Study objectives

1.4.1 Broad Objective

To improve rice seed quality through diversified assessment and identification of seed-borne mycoflora and to evaluate the efficacy of commonly used management practices in Liberia’s agro-ecological zones.

1.4.2 Specific Objectives

1. To assess farmers’ level of awareness on pre- and post-harvest practices linked to seed quality enhancement and deterioration in Liberia.
2. To identify seed-borne fungal pathogens affecting rice seed quality in various agro-ecological zones of Liberia.
3. To determine the efficacy of some botanicals: neem, ginger, and common bean ash, to enhance rice seed quality and longevity in Liberia.
4. To assess in-vitro activities of neem, ginger, garlic and common bean extracts against selected fungal pathogens isolates from seed rice in Liberia

2.0 MATERIALS AND METHODS

2.1 Study location and site characteristics

The study will take place in four (4) of the fifteen (15) counties of Liberia in the rice agro-ecological zones that is, Bong, Lofa, Gbarpolu, Grand, and Gedeh. These counties are located in the Western, Central, Southeastern and Northern Regions of Liberia.

Figure 2: Source: Sampling Area Map in Liberia, retrieved from the world map slightly modified 2020 (unpublished).

2.2 Survey

The survey will be conducted to establish the farmers’ awareness of their seed quality status, seed fungal contamination, source of rice seeds, storage condition stored, and perception towards the indigenous knowledge of seed preservation. Sample rice grain seeds will be sampled from each participant for subsequent analysis. The survey will employ a mixed-structured questionnaire which will be tested from purposefully sampled farmers in each Location.

2.3 Sample size

The population of farmers farming rice in each count will be acquired from the agricultural offices in respective counties.  Systematic random sampling will be used to select participants per county. Microsoft Excel 2019 will be used for the randomization process. The sample size will be calculated as per the equation below.

Eqn 1

Where;

n = sample population per location

µ = total households in the Location growing sweet potatoes

= population of the households in the Location growing sweet potatoes

q = 10% of total households in each Location growing sweet potatoes

Seed Rice Samples

500g of the rice samples will be obtained from each participating farmer. The sample collection will involve a single slotted tube, which will be pierced diagonally through the storage bag. The samples from all sides will be mixed thoroughly on a polythene cloth and a composite of it taken for subsequent analysis.

2.4 Phase 2: Incubation and Examination

Rice grain samples will be incubated for seven (7) days at 20 ⁰C in darkness. At the end of seven days, the seeds will be physically examined for the growth of fungi. Any identified fungi will further be observed under a dissection microscope for fungal structure determination. The fungal growth will be mounted on the microscope slides and observed under-high-power microscope with the magnification of 200 (Mag X 200).

Plating of Seed Rice Samples

Invitro propagation of sample seeds will be conducted using the blotter method (ISTA, 1966; Mathur et al., 2001). grains of rice samples will be plated in a plastic Petri dishes (9.0cm diameter) lined with three (3) moistened blotters in sixteen (16) replicates, making four hundred (400) seeds rice per sample ecological zone as recommended by ISTA (1966).

Incubation of Plated Rice Samples

The plates will be incubated at 28± 2^oC under the 12 hours of alternating cycles of NUV (near-ultraviolet) light provided by Philips black tube and darkness for seven (7) days according to the recommendation of ISTA (1966).

2.5 Phase 3: Efficacy tests with Botanicals

Botanicals Collection and Powder Preparation

The botanical leaves of Neem (Azadirachta indica), roots and barks, typical bean leaves, ginger (Zingiber officinalis) will be harvested 100g, washed thoroughly with hydrochloride and allowed to dry under room temperature. The dried botanicals will be blended in an electrical blender into fine powders and sieved and placed in airtight protective jars separately.  The typical bean leaves will be dried, burned, and the ashes will be put into a tight air jar.

Seed Rice Samples Treatment

In this method, one hundred seed rice will be randomly selected and treated at the various rates of 0.5g, 1g, and 1.5g, respectively. After seven (7) days of incubation in the 20± 2^oC under 12 hours of an alternate of Near Ultra Violet (NUV) light provided by Philips black tubes and darkness for seven (7) days of the recommendation of ISTA (1966) in a completely randomized design, seed rice samples will be tested against botanical powders and synthetic chemical CONCERN-GT-FS-390.

Identification of Fungal Pathogens on the Treated Seed Rice Samples

After the incubation period, the samples will be observed under the stereomicroscope. Where identification becomes challenging, a slide will be prepared temporally for further examinations under the compound microscope, and identification with the aid of results will be recorded according to (Chidambaram et al. 1973).

The objective of this experiment will be to assess in-vitro activities of neem, ginger, garlic, and common bean extracts against selected fungal pathogens isolate from seed rice in Liberia.

2.6 Phase 4: Determination of in vitro inhibitory indices using botanicals

Preparation of Plant Extracts

Aqueous of the botanical’s neem (Azadirachta indica), ginger (Zingiber officinalis), and garlic (Allium sativum) and common bean ash together with synthetic fungicide Benlate will be used in Potato Dextrose Agar (PDA) media to determine their efficacy on inhibiting Aspergillus flavus and Penicillium citinum growth. The method applied will be that of Asare-Bediako et al. (2007) and Shovan et al. (2008). Garlic, ginger, neem, and typical bean leaves will be collected fresh. Each will be washed thoroughly with clean running tap water, then with alcohol (75%) and with six changes of sterilized distilled water (SDW) as described by Rajamanic et al. (2012), except for the common bean leaves will be burned. The ash will be diluted with clean tap water, and the concentration solution will be filtered stored airtight jar. The botanicals will be homogenized with a blender and then poured into tight air bottles. The extracts will be filtered through cheesecloth to obtain aqueous extracts as stock solutions.

Amendment of the Botanical Extracts and the PDA

Potato dextrose agar (PDA) will be prepared as described by the manufacturer. The warm PDA (200 mL) will be amended with one tablet of amoxicillin (200 mg) to check bacterial contamination and then with, 10, 15 and 20 ml of each plant extract to give the three levels of concentrations of 10, 15, and 20% (v/v) of each plant extract. Fungicidal suspensions of different concentrations (10.15 and 20v/v or 100, 200, and 300 ppm) will be prepared by dissolving requisite quantities of Benlate in warm PDA. Approximately 15 ml of each molten PDA amended with the plant extracts, or Benlate suspension will be poured into each 9.0mm Petri-dish and allowed to set.

Plating of Aspergillus Flavus and Penicillium citinum Isolates on the amended PDA

The three-day-old cultures of isolates of A. flavus and P. citinum kept on a slant will be used to inoculate the various PDA plates. Pure cultures will be obtained from the three-day-old A. flavus and P. citinum isolates. Inoculum plugs will be achieved with a sterile cork borer from the growing margin. Pure cultures of A. flavus and P. citinum from the 30 samples will be obtained by transferring fungal colonies to the new PDA plates and incubating them for seven days. The dishes will be wrapped with sterile plastic paper tapes and placed in the incubation room at 28-30⁰C in a Completely Randomized Factorial design with three replications per treatment. Three PDA, Benlate plates will be amended and used as controls in the experiment. The radial diameters of the modified and unamended plates will be measured on the seventh day of the incubated isolates.

The percentage inhibition of A. flavus and P. citinum mycelial growth will be calculated based on the colony diameter on control plates, and fungicide treated plates using the following formula as stated by Sunder and Ishnaveni (1995), with a slight modification:

% inhibition = Y- A * 100

Where,

Y= mycelial growth on the control plate.

A= mycelial growth on the fungicide treated plate.

Statistical Analysis

Survey data will be sorted and subject to descriptive statistics in the form of frequency distribution, line graphs with error bars, and Chi-square correlations using Statistical Package of Service Solutions (SPSS), Version 23 (IBM). Data on the efficiency of botanical herbs in inhibiting fungal growth and enhancing the seed quality will be subjected to the multivariate analysis of variance (MANOVA) and Principal Component Analysis PCA using the GenStat Release 16^th edition.

3.0 EXPECTED RESULTS, BENEFICIARIES, AND POTENTIAL IMPACTS

3.1 Expected Outputs

• The study will give insight into the common rice farmers’ seed quality enhancement strategies in Liberia.
• Documentation of the common fungal rice pathogens in the rice-producing counties of Liberia.
• The study will also provide information on the efficiency of different local botanical herbs in inhibiting fungal pathogens.
  • Study beneficiaries and potential impact

The result will benefit rice value chain stakeholders, including; Rice farmers, by providing economically feasible techniques of ensuring seed quality. The study result is anticipated to reduce contribute to improved local rice production hence reducing the government expenditures on importations. Documentation of information on the local practices regarding seed quality maintenance is deemed to spearhead the development of more accurate and practical policies towards the country’s actualization of food and nutritional security.

APPENDIX

Appendix 1: Work plan

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