Peanut (Arachis hypogaea L.) is one of the most widely grown legumes by smallholder farmers in Malawi (Nyondo et al., 2018). Peanut is a relatively inexpensive source of dietary protein and other essential nutrients for both urban and rural households (Okello et al., 2010a 2010b). Peanut also serves as an important source of livestock and poultry feed (Kochhar 1986; Usman et al., 2012). However, yield is low (700 kg/ha) in Malawi compared to other countries because most smallholder farmers in Malawi grow peanut with little or no inputs (Ngwira et al., 2019). In addition to low yields, aflatoxin, a mycotoxin produced by Aspergillus flavus and A. parasiticus contaminates peanut-based food products and contributes to poor health (Bowen and Hagan, 2015). Peanut and food products that contain aflatoxin above established levels can limit marketing opportunities (FAO, 2001; Matumba et al., 2015; Waliyar et al., 2010). The European Union and Malawi Bureau of Standards accepts aflatoxin level of 4 μg/kg while the World Health Organization (WHO) standard is 20 μg/kg (Monyo et al., 2012; Otsuki and Wilson, 2001). Aflatoxin contamination adversely affects human health in a number of ways including liver cancer and immunosuppressive effects (Guchi, 2015; WHO, 2006).

Aflatoxin can be present prior to harvest during the growing cycle when peanut is exposed to prolonged high day and night temperatures and drought during pod filling (Payne, 1998; Sanders et al., 1984). Pre-harvest aflatoxin contamination in peanut is often associated with cracked pods that allow entry of soil that contains A. flavus (Craufurd et al., 2006). High relative humidity and soil temperatures ranging from 25 to 35 C are favorable for A. flavus development (Bowen and Hagan, 2015; Cole et al., 1984; Hill et al., 1983). Weather conditions that result in delayed harvest increase the likelihood of greater damage from arthropods and vertebrates that increase pod damage and subsequent movement of soil into pods (Desai et al., 2008). Minimizing stress associated with high temperatures, drought, and damage from other organisms can minimize aflatoxin contamination of peanut going into drying and storing steps in the supply chain (Torres et al., 2014).

Establishing adequate populations of peanut increases yield and uniformity of harvested peanut (Onat et al., 2017; Okello et al., 2010a 2010b). The current recommended density of peanut planted in Malawi is 89,000 plants/ha when established in rows spaced 75 cm apart at a distance of 15 cm between plants in each row (Ngwira et al., 2019). However, Onat et al. (2017) reported that increasing the population to 133,000 plants/ha increased yield compared with lower densities in Turkey. Greater yields were also noted in the United States (Kvien et al., 1987) and India (Rasekh et al., 2010) when higher plant populations were established as compared with lower plant populations. As farmers consider increasing seeding rates to achieve higher plant populations, it is important to determine if this approach affects aflatoxin contamination. The impact of plant population on aflatoxin contamination has not been documented in the peer-reviewed literature.

Harvesting peanut when the kernels and pods reach physiological maturity often results in greater yields, higher market grade characteristics (Okello et al., 2010a 2010b), and can minimize seed infection by A. flavus (Mehan et al., 1986; Sanders et al., 1985). Harvesting peanut prior to physiological maturity can result in a distribution of kernels that contain a higher percentage of smaller and less mature kernels as compared with harvesting at physiological maturity (Carter et al., 2017). When peanut is harvested past physiological maturity, pods have greater exposure to pathogens and arthropods that can cause damage (Okello et al., 2010a 2010b; Singh and Oswalt, 1995). Pods that are past physiological maturity for an extended period of time, especially if soil moisture is adequate, can sprout and result in lower yield and quality (Nautiyal et al., 2001; Singh and Oswalt, 1995). Pods can also shed from plants and may not be harvestable (Kaba, 2014).

The majority of farmers in Malawi establish plant populations lower than what is needed to optimize yield because of seed quality issues and expense (Ngwira et al., 2019). Farmers also do not fully appreciate the relationship of harvest date, physiological maturity, and the impact of pests on yield and quality, including the human health and marketing ramifications of contamination with aflatoxin (Kaba, 2014). The relationship between plant population and harvest date has not been evaluated in Malawi in a systematic way with respect to peanut yield and aflatoxin contamination. Therefore, research was conducted to determine the impact of plant population and harvest date on peanut yield and contamination by aflatoxin at harvest in Malawi.

The experiment was conducted at Mpatsanjoka farm in Salima district of Malawi (13°42.740′S, 034°28.879′E) during crop cycles in 2015-2016 and 2016-2017. Mpatsanjoka farm is located near Lake Malawi at an elevation of 550 m above sea level. Monthly average rainfall from Nov through March for each cropping cycle is presented in Table 1. No measurable rainfall was observed after March of each year. Soil was a sandy loam with pH 5 and organic matter content of 2.7%. Peanut was seeded on 13 December 2015 and 22 December 2016 in raised seedbeds with a height of 20 to 30 cm (Table 2). Immediately after planting, dimethenamid-P (Frontier-P herbicide, Australian Pesticides and Veterinary Medicine Authority, Kingston, Australia) was applied at 0.84 kg ai/ha to control weeds. Hand weeding was used to throughout the season to minimize weed interference. No other pesticides were applied to control arthropods or pathogens.

Treatments consisted of the virginia market type cultivar CG7 (Ngwira et al., 2019) seeded in single, twin, and triple row patterns at rates of 89,000, 178,000, and 285,000 seed/ha to establish low, medium, and high plant populations, respectively (Table 3). The seeding rate of 89,000 seeds/ha was established on beds spaced 75 cm apart with one row of peanut with 15 cm between seeds. The seeding rate of 178,000 seeds/ha was established on beds with 75-cm centers with two rows of peanut spaced 18 cm apart with 15 cm between seeds. The seeding rate of 285,000 plants/ha was established on beds with 75-cm centers with three rows of peanut spaced at 18 cm with 7 cm between seeds. The final in-row spacings for these respective plant populations/ha during each cycle are presented in Table 3. Seeds were treated with thiram (Thiram 50WP, Bayer Crop Science, Research Triangle Park, NC) at 3g/kg of peanut seed and planted in furrows created manually at a depth of 3 cm. Within each plant population, peanut was harvested 10 d before physiological maturity, at physiological maturity, 4 wk after physiological maturity, and 6 wk after physiological maturity. The internal color of pods was used to determine maturity by collecting representative plants across the field. When 70% of pods visible darkening caused by removal of the endocarp as kernels increased in size and development, peanut was considered physiologically mature (Ngwira et al., 2019). Approximately 50% of pods expressed this level of darkening 10 d prior to physiological maturity. After gently removing peanut pods from the soil, above-ground vegetation, pods, and remaining roots were placed on a Mandela cock (Ngwira et al., 2019). When pod moisture was 8.5%, total plant biomass, pod weight, grain weight, and aflatoxin contamination in pods were determined.

Aflatoxin concentration was determined using 300 g of shelled peanut from each plot. The sample was weighed and homogenized using a hand grinder (Globe Trek, Navi Mumbai, Mumbai) and thoroughly mixed. A 10-g sub-sample was removed and agitated in 30 ml of ethanol (65%) and water (35%) for 1 min. The sub-sample was filtered using a 500 μL pipette (Fisher Scientific, Pittsburg, PA) with the diluent. One hundred μL of the filtered liquid was inserted into the sampling cup and left in the cup for 6 min. Neogen Reveal Q+ lateral flow strips (Neogen Corp., Lansing, MI) were inserted into the strip holder (Neogen Corp., Lansing, MI) to determine aflatoxin concentration using the Mobile Assay mReader software (Mobile Assay Inc., Boulder, CO).

Data for total biomass, pod weight, grain weight, and aflatoxin contamination were subjected to analysis of variance for the split-plot design using GENSTAT 18^th edition computer package (VSN international, England, United Kingdom). Seeding density served as the whole plot unit and harvest date served as the sub-plot unit. Differences of main effects and interactions were separated using Fisher’s Protected LSD test at p < 0.05.

Interactions of growing cycle by seeding rate by harvest date were not significant for plant biomass at harvest, pod yield, and aflatoxin concentration (p > 0.05). However, main effects of seeding rate and harvest date, the interaction of growing cycle and seeding rate, and the interaction of growing cycle and harvest date were significant. Data for plant biomass, pod yield, and aflatoxin concentration will be presented for these interactions combined over growing cycle and the other treatment factor (Tables 4 and 5). Oakes et al. (2020) reported that both seeding rate and harvest date affected pod yield but response to these treatment factors was independent.

Above-ground plant biomass increased as seeding rate and subsequent plant population increased in one of the two growing cycles (Table 4). In 2016-2017 growing cycle, plant biomass increased for each increase in plant density. Bell et al., (1987) reported that peanut biomass increased from 12,600 kg/ha to 16,900 kg/ha with increasing plant density up to the maximum density of 588,000 plants/ha.

Peanut pod yield was greater in both growing cycles for the seeding rate of 285,000 seed/ha compared with the lower seeding rates (Table 4). In the 2015-2016 growing cycle, no difference in yield was observed for the low and medium seeding rates. However, in the 2016-2017 growing cycle, peanut yield for the medium seeding rate exceeded that of the low seeding rate. Aflatoxin contamination at harvest was not affected by seeding rate in either growing cycle.

Increasing the seeding rate often ensures greater uniformity of pod maturity, improved quality of grain for marketing, and maximum yield (Okello et al., 2010a 2010b). However, variation in response to seed spacing has been observed. Konlan et al. (2013) in Ghana reported that decreasing spacing between seeds increased yield by 6.2% in one years and 16.0% in a second year of the study. Rasekh et al. (2010) reported higher yields when the in-row seeding rate was increased from 3 plant/m to 8.3 plants/m. Awal and Aktar (2015) and Gabisa et al. (2017) reported greater yield with a plant population of was increased. El Naim et al. (2011) reported that peanut plants spaced 10 cm apart yielded 40% less than peanut planted 40 cm apart under rain-fed conditions.

Above-ground peanut biomass was greater 10 d prior to physiological maturity compared to mass at physiological maturity (Table 5). When harvest was delayed past physiological maturity, biomass continued to decrease. This was due to significant vegetative growth and by this time the crop did not lose its leaves while the soil was still moist. Peanut pod yield was similar when harvested at physiological maturity, or 10 d prior to physiological maturity (Table 5). In the first growing cycle, yield was similar when peanut was harvested at physiological maturity or 4 wk after physiological maturity. In contrast, in the second growing cycle, peanut yield was lower when harvested 4 wk after physiological maturity than harvest at physiological maturity. Delaying harvest to 6 wk after physiological maturity resulted in the lowest grain yield. The decrease in pod yield with the delayed harvesting was due to field losses as many remaining in the field due to natural pod shed. Young et al. (1982) estimated typical digging losses of 8% of total yield, but can reach 40% at dates when harvest is delayed past optimal maturity. Okello et al. (2010a 2010b) reported that delayed harvesting causes yield losses of greater than 400 kg/ha and kernel quality reduced by 3%.

Aflatoxin contamination was similar when peanut was harvested 10 d prior to physiological maturity or at physiological maturity (Table 5). In the first growing cycle, aflatoxin contamination was greater when harvest was delayed by 4 and 6 wk past physiological maturity compared with harvest 10 d prior to physiological maturity or at physiological maturity. By 4 and 6 wk after physiological maturity, aflatoxin contamination was 40.0 to 48.6 μg/kg. During the second growing cycle, aflatoxin contamination did not exceed 13.6 μ/kg. The difference in aflatoxin due to harvest date most likely was associated with rainfall patterns during the latter part of the growing cycle in March. Rainfall ceased during the 2015-2016 growing cycle in March while rainfall during March 2017 was during 264 mm and just under twice the total rainfall during the growing cycle (Table 1). Adequate rainfall which reduces aflatoxin contamination in peanut because it increases tissue integrity hence reduce invasion of A. flavus (Diao et al., 2015). The lower concentration of aflatoxin most likely was associated with complete maturation of pods when rainfall was more abundant in March which minimized infection when soil moisture in soil was higher. Sanders et al. (1984) reported that peanut pods developed without damage to shells and limit entry of Aspergillus spp. to colonize pods. Okello et al. (2010a 2010b) reported that harvesting peanut at optimum maturity reduces incidences of aflatoxin contamination. Peanut harvested after physiological maturity had high aflatoxin contamination because of over maturity and delayed harvesting which increases aflatoxin contamination (Diener and Davis, 1977; Okello et al., 2010a 2010b).

In summary, increasing seeding rates and subsequent plant populations up to 285,000 seed/ha resulted in the greatest yield across the two growing cycles. However, seeding rate had no effect on aflatoxin contamination at harvest. It is postulated that higher plant populations likely shade soil and create cooler soil environment that is less conducive to A. flavus growth and infection. However, our results did not support that hypothesis. It was also postulated that harvesting peanut prior to physiological maturity would result in less aflatoxin compared with harvesting at physiological maturity or past physiological maturity. Our results are in contrast to those by Bowen and Hagan (2015) who reported less aflatoxin when peanut was harvested earlier than recommended to optimize pod yield when conditions were favorable for A. flavus development. However, our results were consistent with those of Young et al., (1982) demonstrating that delaying harvest past physiological maturity can result in lower yield due to leaf and pod shed due to disease and natural processes and sprouting of seed or damage caused by arthropods in soil. Pod damage caused by arthropods can allow soil to enter pods and increase the amount of A. flavus in pods and subsequently cause greater aflatoxin contamination.

This is the first experiment in the peer-reviewed literature that has addressed the impact of seeding rate and subsequent contamination by aflatoxin. Our results indicate that plant population and harvest date do not interact for pod yield or aflatoxin contamination. Oakes et al. (2020) also reported that both seeding rate and harvest date affected peanut pod yield independently. It is important to note that plant populations were established using three different planting patterns with peanut plants distributed differently across beds. Additional research is needed to determine the impact of a more dense population using a similar planting pattern to determine the impact of plant population on yield and aflatoxin contamination.