GENETIC ANALYSIS OF AFLATOXIN AND AGRONOMIC TRAITS IN GROUNDNUT (ARACHIS HYPOGAEA L

ABSTRACT
Groundnut (Arachis hypogaea L.) is one of the major crops susceptible to Aspergillus flavus infection and subsequent aflatoxin contamination which adversely affects its production and utilization in the small holder setting. Although there are several management strategies that may reduce aflatoxin contamination of groundnuts, the pre-eminent strategy for the prevention of aflatoxin is to develop host resistance to A. flavus. The objectives of this study were to determine the level of resistance to A. flavus and aflatoxin accumulation among selected groundnut genotypes, to assess the mode of gene action controlling resistance to aflatoxin accumulation and agronomic traits and to estimate heritability for resistance to A. flavus and aflatoxin accumulation. Eight groundnut varieties comprising of three A. flavus resistant lines and five susceptible varieties were obtained from Institute for Agricultural Research, Samaru. The genotypes were crossed in a 3 x 5 North Carolina (NC II) design II fashion to generate 15 F₁ hybrids. The F₁ seeds were selfed to F₂ and then evaluated alongside the eight parents for twelve quantitative characters using randomised complete block design (RCBD) with three replications at the Department of Plant Science screen-house under artificial A. flavus inoculation. Analysis of variance revealed significant (P≤0.05) differences among the groundnut genotypes for all traits except for 100-kernel weight and shelling percentage. The mean performance of the parents showed that SAMNUT 22 (2.50 ng / g ), ICGV 91317 (6.31 ng / g ) and ICGV
 
91324  (12.23 ng / g )  recorded  lowest  aflatoxin  accumulation  value  of  less  than
 
20.00 ng / g  and the crosses: SAMNUT 26 x ICGV 91317 (0.8 ng / g ), SAMNUT 26
 
x ICGV 91328 (1.2 ng / g ), SAMNUT 25 x ICGV 91324 (3.92ng/g), SAMNUT 22 x
 
ICGV 91328 (4.4 ng / g ) and SAMNUT 22 x ICGV 91324 (12.3 ng / g ), as the best
for resistance to A. flavus infection and aflatoxin accumulation under the United States Food and Drug Administration, which sets the limit of aflatoxin in food and feeds at 20 ng/g. The general (GCA) and specific (SCA) combining abilities were significant (P≤0.05) for most characters indicating the role of both additive and non-additive gene effects in the expression of most characters. High broad-sense heritability values were obtained for days to 50% flowering (88.9%), plant height (84.0%), haulm weight (86.8%), kernel infection (85.3%) and aflatoxin accumulation
 
(99.7%). The parental genotypes, SAMNUT 23, SAMNUT 24, SAMNUT 25, SAMNUT 26, ICGV 91324, ICGV 91328 were good general combiners for aflatoxin B₁ accumulation. Among the progenies, SAMNUT 22 x ICGV 91324 and SAMNUT 23 x ICGV 91317 with positive SCA effects for haulm weight and negative SCA effects for kernel infection and aflatoxin accumulation. A. flavus infection among the parents and progenies correlates poorly to aflatoxin contamination. The study revealed that non-additive gene action was more important than additive gene action for resistance to A. flavus infection, while additive gene action was more predominant for resistance to aflatoxin accumulation. The information provided from this study could be utilized in planning breeding programme for the development of groundnuts with improved pod yield, haulm yield, resistance to A. flavus infection and aflatoxin accumulation.
CHAPTER ONE
 
 
1     Introduction
 
 
Groundnut (Arachis hypogaea L.) is an annual legume which is also known as peanut. It is the 13^th most important food crop of the world. Today, groundnut is the world’s 4th most important source of edible oil and the 3^rd most important source of vegetable protein in the world. Groundnut is cultivated in more than 100 countries in 6 continents and covers an area of 26.4 million hectares worldwide with a total production of 41.3 million metric tons, and an average productivity of 1.4 metric tons per hectare (FAO, 2014). Major groundnut producers in the world are: China, India, Nigeria, USA, Indonesia and Sudan. Developing countries account for 96% of the global groundnut area and 92% of the global production (FAO, 2014). Groundnut kernels are consumed directly as raw, roasted or boiled kernels or oil extracted from the kernel is used as culinary oil. Groundnut seeds contain high quality oil (50%), easily digestible protein (25%) and carbohydrates (20%). It is also used as animal feed and industrial raw material. These multiple uses of groundnut make it an excellent cash crop for domestic markets as well as for foreign trade in several developing and developed countries (Pandey et al., 2012).
Problem statement
Groundnut is affected by several diseases like leaf spots, collar rot, rust, bud necrosis and stem necrosis (Prasada et al., 2012). Apart from these, aflatoxin contamination is one of the major problems, produced in the infected groundnut seeds by Aspergillus flavus Link ex fries and Aspergillus parasiticus Speare, particularly at the end of
season under drought conditions (Fountain et al., 2014). Aflatoxins, especially the most potent aflatoxin B₁, are secondary metabolite produced by A. flavus. They are highly carcinogenic, immunosuppressive agents, highly toxic and fatal to humans and animals particularly affecting liver and digestive track (Wild and Gong 2010). These health problems are more severe in African communities due to exposure to aflatoxins throughout their lives (Wild and Gong 2010). There have been increasing reports of aflatoxin contamination in freshly harvested groundnuts in several countries of sub-Saharan Africa. Recently, 22–54% of groundnut samples collected during 2009 and 2010 from different groundnut growing areas in Nigeria showed >20 ng g⁻¹ of aflatoxins (Waliyar et al., 2016). This contamination renders the commodity unfit for human consumption and unacceptable for trade in high-value markets. Therefore, aflatoxin contamination in groundnut as well as in maize has been considered as a major non-tariff barrier to international trade since agricultural products that exceed the permissible levels of contamination (4 to 20 ng/g) are banned. About $1.2 billion in commerce is lost annually due to aflatoxin contamination, with African economies losing $450 million each year (IITA, 2013).
Following the outbreak of aflatoxicosis and the enormous economic loss in the poultry industry of Britain in 1961, the Federal government of Nigeria, in order to protect her export trade initiated screening studies to determine the extent of aflatoxin contamination of groundnut and groundnut products (Halliday and Kazaure, 1967). McDonald and Harkness (1965) found aflatoxins in groundnut samples from Zaria, Kano and Mokwa, in Northern Nigeria. Bassir (1969) isolated aflatoxin B₁ from various mouldy food materials offered for sale in Ibadan markets. Since then, toxigenic fungi and mycotoxins have been found in various foods and feedstuffs in many regions of Nigeria. Thus, mycotoxigenic fungi belonging to not less than forty
five fungal genera and about twenty different mycotoxins have been detected in Nigerian foods and foodstuffs (Ezekiel et al., 2012). Nigeria has experienced high recorded aflatoxin exposure levels in humans and has also reported the highest estimated number of cases of hepatocellular carcinoma (HCC-liver cancer) attributable to aflatoxins in the whole world (Liu and Wu, 2010). Due to these health risks, many countries have established strict regulations regarding the permissible levels of aflatoxins in food and feed. For the Nigeria, the limit for all foods is 20 ng/g of total aflatoxins (B₁, B₂, G₁ and G₂) except milk, which has a limit of 0.5 ng/g of aflatoxin M₁ (Ezekiel et al., 2012).
Justification
In Nigeria, groundnut farmers rely on cultural practices (such as irrigation, early planting, rapid drying after harvest) and lately bio-controls to manage A. flavus infection and to reduce aflatoxin contamination. Irrigation has been shown to reduce preharvest aflatoxin contamination in groundnut and maize (Waliyar et al., 2015). The biocontrol strategy through the application of atoxigenic strains of A. flavus to compete with toxigenic A. flavus in the field has been shown to significantly reduce aflatoxin contamination in the African countries (Probst et al., 2011). In general, good cultural and management practices as well as biocontrol can reduce, but not eliminate preharvest aflatoxin contamination as some of the management practices are not always available or cost-effective (Waliyar et al., 2015). Therefore, there was a major effort to identify germplasm with natural resistance to A. flavus infection (preharvest resistance) with diminished accumulation of aflatoxin in the past four decades. Attempts have identified potentially resistant groundnut genotypes for pre-harvest aflatoxin contamination (Anderson et al., 1995; Upadhyaya et al., 2004; Waliyar et
al., 2016). Three groundnut genotypes such as ICGV 87084, ICGV 87094 and ICGV 87110 were identified to be resistant to A. flavus and aflatoxin contamination as evaluated in Niger, Senegal and Burkina Faso in West Africa (Waliyar et al., 1994). Further improved groundnut germplasm lines such as ICGV 91278, ICGV 91283 and ICGV 91284 were registered as resistant to A. flavus seed infection (Upadhyaya et al., 2000). However, undesirable agronomic characteristics such as poor shelling outturn and late maturity (Upadhyaya et al., 2004) associated with these genotypes, hinder their direct use as commercial aflatoxin resistant groundnut varieties in Nigeria. The effort to incorporate resistant traits from these germplasm into commercial background has been a challenge due to lack of a quick, inexpensive, and reliable means to evaluate resistance (Moreno and Kang, 1999), and a poor understanding of host resistance mechanisms. Genetic improvement of quantitative characters in groundnut through different breeding programs desires the information on the nature and magnitude of gene effects. The genetic potential of groundnut can be predicted and measured by the estimates of genetic effects. Based on this, various breeding strategies can be formulated towards the genetic improvement for resistance to A. flavus infection, aflatoxin accumulation and other agronomic characters. It is in the light of the above that this study was undertaken with the following objectives:

1. To determine the level of resistance to Aspergillus flavus and aflatoxin accumulation among selected groundnut genotypes and their F₂ progenies
2. To assess the mode of gene action controlling resistance to kernel infection by flavus and agronomic trait

• To estimate heritability for resistance to flavus and aflatoxin accumulation