Growth and Toxigenicity of A. flavus on Resistant and Susceptible Peanut Genotypes

Aflatoxin contamination poses serious health concerns to consumers of peanut and peanut products. This study aimed at investigating the response of peanuts to Aspergillus flavus infection and aflatoxin accumulation. Isolates of A. flavus were characterised either as aflatoxigenic or non-aflatoxigenic using multiple cultural techniques. The selected isolates were used in an in vitro seed colonisation (IVSC) experiment on two A. flavus-resistant and susceptible peanut genotypes. Disease incidence, severity, and aflatoxin accumulation were measured. Genotypes differed significantly (p < 0.001) in terms of the incidence and severity of aflatoxigenic and non-aflatoxigenic A. flavus infection with the non-aflatoxigenic isolate having significantly higher incidence and severity values. There was no accumulation of aflatoxins in peanut genotypes inoculated with non-aflatoxigenic isolate, indicating its potential as a biocontrol agent. Inoculations with the aflatoxigenic isolate resulted in the accumulation of aflatoxin B1 and G1 in all the peanut genotypes. Aflatoxin B2 was not detected in ICGV–03401 (resistant genotype), while it was present and higher in Manipinta (susceptible genotype) than L027B (resistant genotype). ICGV–03401 can resist fungal infection and aflatoxin accumulation than L027B and Manipinta. Non-aflatoxigenic isolate detected in this study could further be investigated as a biocontrol agent.

Peanut production is faced with challenges such as inadequate inputs, unreliable rains, the use of unimproved varieties, and disease and pest infestation [9]. In addition to the above, peanut is reported to be prone to Aspergillus species infection [10][11][12], which leads to a decline in its quality and quantity [13,14]. Among the Aspergillus species, A. flavus and A. parasiticus are Additionally, the isolates of A. flavus on PDA in each Petri dish were exposed to 2 mL of concentrated Ammonia solution for 4 to 5 min ( Figure 2). Additionally, the isolates of A. flavus on PDA in each Petri dish were exposed to 2 mL of concentrated Ammonia solution for 4 to 5 min ( Figure 2). Additionally, the isolates of A. flavus on PDA in each Petri dish were exposed to 2 mL of concentrated Ammonia solution for 4 to 5 min ( Figure 2). The results revealed that, on the PDA, 62.5% were characterised as aflatoxigenic whilst 37% were non-aflatoxigenic A. flavus. Similarly, on the YESA+β-cyclodextrin, The results revealed that, on the PDA, 62.5% were characterised as aflatoxigenic whilst 37% were non-aflatoxigenic A. flavus. Similarly, on the YESA+β-cyclodextrin, 81.25% were aflatoxigenic while 18.75% were non-aflatoxigenic (Table 1). Additionally, it was observed that 13 out of 16 A. flavus isolates, which represents 81.25%, were aflatoxigenic while 3 isolates (18.75%) were non-aflatoxigenic when A. flavus culture plates were exposed to concentrated ammonia solution (Table 1).

Morphological Variation in the Growth of Aflatoxigenic and Non-Aflatoxigenic A. flavus
The results on the radial growth of aflatoxigenic isolate 8 (A8) and isolate 2 (A2), and non-aflatoxigenic A. flavus isolate 11 (NA11) and isolate 12 (NA12) revealed that A. flavus increased in diameter on the PDA as the days progressed, as shown in (Figure 3). The radial growth of the non-aflatoxigenic A. flavus isolates were higher than the aflatoxigenic A. flavus isolates. Additionally, the area under the disease progress curve (AUDPC) values of aflatoxigenic A. flavus isolates (A2 and A8) were significantly lower than AUDPC values for non-aflatoxigenic A. flavus isolates (NA11 and NA12) during the 8-day incubation period (Figure 4).

Incidence of Aflatoxigenic and Non-Aflatoxigenic A. flavus
There was highly significant (p < 0.001) variation in incidence of the three peanut genotypes (ICGV−03401, L027B, and Manipinta) inoculated with aflatoxigenic and nonaflatoxigenic A. flavus. The resistant genotypes (ICGV−03401 and L027B) had incidence values less than 40% for aflatoxigenic and non-aflatoxigenic A. flavus whereas the sus-

Incidence of Aflatoxigenic and Non-Aflatoxigenic A. flavus
There was highly significant (p < 0.001) variation in incidence of the three peanut genotypes (ICGV-03401, L027B, and Manipinta) inoculated with aflatoxigenic and nonaflatoxigenic A. flavus. The resistant genotypes (ICGV-03401 and L027B) had incidence values less than 40% for aflatoxigenic and non-aflatoxigenic A. flavus whereas the susceptible genotype (Manipinta) had the highest (78%-80%). However, the percentage incidence of non-aflatoxigenic A. flavus on all the genotypes was higher than the percentage incidence of aflatoxigenic A. flavus ( Figure 5). ceptible genotype (Manipinta) had the highest (78%-80%). However, the percentage incidence of non-aflatoxigenic A. flavus on all the genotypes was higher than the percentage incidence of aflatoxigenic A. flavus ( Figure 5).

Severity of Aflatoxigenic and Non-Aflatoxigenic A. flavus
There were significant (p < 0.001) differences in severity of the three peanut genotypes inoculated with aflatoxigenic and non-aflatoxigenic A. flavus. The resistant genotypes (ICGV−03401 and L027B) had severities of less than 30% for aflatoxigenic and nonaflatoxigenic A. flavus whereas the susceptible genotype (Manipinta) had the highest (53%-63%). However, the incidence of non-aflatoxigenic A. flavus was higher in all the peanut genotypes than aflatoxigenic A. flavus ( Figure 6).

Severity of Aflatoxigenic and Non-Aflatoxigenic A. flavus
There were significant (p < 0.001) differences in severity of the three peanut genotypes inoculated with aflatoxigenic and non-aflatoxigenic A. flavus. The resistant genotypes (ICGV-03401 and L027B) had severities of less than 30% for aflatoxigenic and nonaflatoxigenic A. flavus whereas the susceptible genotype (Manipinta) had the highest (53%-63%). However, the incidence of non-aflatoxigenic A. flavus was higher in all the peanut genotypes than aflatoxigenic A. flavus ( Figure 6).

Quantification of Aflatoxin
Extraction and estimation of aflatoxin accumulation in aflatoxigenic and nonaflatoxigenic isolates in peanut was quantified using HPLC ( Figure 7) with aflatoxin standards ( Figure 8). The chromatogram results revealed the peaks at retention times of about 3 min. Figure 7A,C confirmed the detection and accumulation of aflatoxin B1, B2, and G1 in L027B and Manipinta, while only B1 and G1 was detected in ICGV−03402 Figure 7B following the inoculation of peanut seeds with the aflatoxigenic isolate A2. However, chromatogram in Figure 7D indicates non-detection of aflatoxins in peanut genotypes L027B, ICGV−03401, and Manipinta by non-aflatoxigenic isolate used in inoculation. Aflatoxin accumulation of the three peanut genotypes (ICGV−03401, L027B, and Manipinta) based on HPLC results is shown in Table 2.

Quantification of Aflatoxin
Extraction and estimation of aflatoxin accumulation in aflatoxigenic and non-aflatoxigenic isolates in peanut was quantified using HPLC ( Figure 7) with aflatoxin standards (Figure 8). The chromatogram results revealed the peaks at retention times of about 3 min. Figure 7A,C confirmed the detection and accumulation of aflatoxin B 1 , B 2 , and G 1 in L027B and Manipinta, while only B 1 and G 1 was detected in ICGV−03402 Figure 7B following the inoculation of peanut seeds with the aflatoxigenic isolate A2. However, chromatogram in Figure 7D indicates non-detection of aflatoxins in peanut genotypes L027B, ICGV-03401, and Manipinta by nonaflatoxigenic isolate used in inoculation. Aflatoxin accumulation of the three peanut genotypes (ICGV-03401, L027B, and Manipinta) based on HPLC results is shown in Table 2.

Discussion
Several interventions have been reviewed and recommended for aflatoxin agement in peanuts; among these, host plant resistance have proven to be the most tive [29]. The use of resistant genotypes if available is the best option. Apart from less cost-effective and easy to disseminate, it does not require any special expertise application and it is compatible with other control methods [29,30]. An understandi the genetics and mechanisms of resistance to aflatoxin will help efficiently breed f sistance to this trait in peanut.
In order to elucidate the mechanisms of resistance in selected A. flavus resistan susceptible peanut genotypes, it was hypothesised that (1) Non-aflatoxigenic A. grows faster than aflatoxigenic A. flavus and could therefore limit the accumulation latoxin and (2) A. flavus infection and colonisation is influenced by the genetic ma of the peanut genotype and that subsequent aflatoxin production is dependent o toxigenicity of the pathogen. These were investigated through multiple cultural me of determining the aflatoxigenicity of the isolates followed by a radial growth bio to determine which of the isolates was fast growing. An in vitro seed colonisation (I assay was then conducted with the selected non-aflatoxigenic isolate and aflatoxi isolate, followed by detection and quantification of aflatoxins produced and accum ed in the infected tissues.
A. flavus was identified by its characteristic yellow green colour and with cir conidia which had a whitish colour around the edge [31].
The present study revealed that under the UV light observations, 81.25% of th lates were detected by YESA+β-cyclodextrin medium as aflatoxigenic, whereas on medium, 62.5% of the isolates were aflatoxigenic. A similar observation was mad Mahmoud et al. [32], who detected aflatoxigenic isolates under UV light using amended with Sodium Chloride and YESA and the positive isolates observed were and 53.33% respectively. This gives an indication that different culture media hav ferent abilities in detecting aflatoxigenic and non-aflatoxigenic A. flavus isolates.

Discussion
Several interventions have been reviewed and recommended for aflatoxin management in peanuts; among these, host plant resistance have proven to be the most effective [29]. The use of resistant genotypes if available is the best option. Apart from being less costeffective and easy to disseminate, it does not require any special expertise in its application and it is compatible with other control methods [29,30]. An understanding of the genetics and mechanisms of resistance to aflatoxin will help efficiently breed for resistance to this trait in peanut.
In order to elucidate the mechanisms of resistance in selected A. flavus resistant and susceptible peanut genotypes, it was hypothesised that (1) Non-aflatoxigenic A. flavus grows faster than aflatoxigenic A. flavus and could therefore limit the accumulation of aflatoxin and (2) A. flavus infection and colonisation is influenced by the genetic makeup of the peanut genotype and that subsequent aflatoxin production is dependent on the toxigenicity of the pathogen. These were investigated through multiple cultural methods of determining the aflatoxigenicity of the isolates followed by a radial growth bioassay to determine which of the isolates was fast growing. An in vitro seed colonisation (IVSC) assay was then conducted with the selected non-aflatoxigenic isolate and aflatoxigenic isolate, followed by detection and quantification of aflatoxins produced and accumulated in the infected tissues.
A. flavus was identified by its characteristic yellow green colour and with circular conidia which had a whitish colour around the edge [31].
The present study revealed that under the UV light observations, 81.25% of the isolates were detected by YESA+β-cyclodextrin medium as aflatoxigenic, whereas on PDA medium, 62.5% of the isolates were aflatoxigenic. A similar observation was made by Mahmoud et al. [32], who detected aflatoxigenic isolates under UV light using PDA amended with Sodium Chloride and YESA and the positive isolates observed were 20% and 53.33% respectively. This gives an indication that different culture media have different abilities in detecting aflatoxigenic and non-aflatoxigenic A. flavus isolates.
Again, by exposing the isolates to concentrated ammonia vapour, the study revealed that 13 (81.25%) out of the 16 isolates were observed to be aflatoxigenic whereas the remaining 3 (18.75%) isolates were non-aflatoxigenic A. flavus. Similar observations were made by Navya et al. [13], who identified 81.58% (31 out of 38 A. flavus isolates) to be aflatoxigenic A. flavus while the remaining 7 (18.42%) were non-aflatoxigenic in A. flavus culture plates exposed to ammonia vapour. This trend may mean majority of A. flavus isolates in most areas are aflatoxigenic as reported in previous studies [33][34][35]. The percentage of aflatoxigenic strains of A. flavus has been demonstrated to vary with the kind of substrate and environmental conditions, according to a study by Bharose et al. [19].
The AUDPC values and morphological variation in radial growth revealed that the non-aflatoxigenic A. flavus isolates grows faster than the aflatoxigenic isolates. This corroborates with reports that in nature the non-aflatoxigenic A. flavus grows faster than aflatoxigenic A. flavus since it does not require energy to produce aflatoxins and can invade and displace aflatoxigenic isolates [13,35].
The peanut seeds from the three genotypes reacted differently to aflatoxigenic and non-aflatoxigenic A. flavus infection as shown by their incidence and severity values. These differences could be associated with the differences in genetic makeup of the genotypes. ICGV-03401 and L027B had the lowest incidence and severity values, Manipinta had the highest. Among the two putative resistant lines, ICGV-03401 highly restricted fungal infection and colonisation, which could be attributed to the thickness and permeability of its seed coat [20,29,36] or seed coat biochemicals [37].
The selected aflatoxigenic and non-aflatoxigenic isolates infected and colonised the peanut genotypes with the non-aflatoxigenic isolate, resulting in the highest percentage incidence and severity. This means that the genotypes were not immune to neither aflatoxigenic nor non-aflatoxigenic A. flavus infection since both strains have an equal chance to survive and multiply. The genotypes also followed the same pattern (ICGV-03401 > L027B > Manipinta) in terms of their resistance to either aflatoxigenic or non-aflatoxigenic isolates. HPLC results, however, revealed that inoculation with a non-aflatoxigenic isolate did not result in the production and accumulation of aflatoxin but aflatoxins were detected in the genotypes inoculated with the aflatoxigenic strain. Resistance mechanisms to A. flavus infection are the same irrespective of the toxigenicity of the isolate. In terms of aflatoxin production, a pathogen factor (aflatoxigenic) and genotype factor (susceptibility) is involved. The toxigenicity of the isolate is very important in the production of aflatoxin. Peanut genotypes that exhibit strong resistance to any of these strains through IVSC may subsequently be resistant to aflatoxin production. IVSC could therefore be regarded as a quick way of screening for resistance to A. flavus infection and aflatoxin accumulation.
Aflatoxin types detected and identified in the three peanut genotypes were AFB 1 , AFB 2 , and AFG 1 with aflatoxin accumulation ranging from 0.4 ppb to 2.096 ppb. These values were obtained after 7 days incubation period following inoculation of a highly concentrated toxigenic strain. Increase in incubation period does not really influence aflatoxin concentration [38]; however, these values could increase with respect to favourable environmental factors such as temperature and water activity and storage methods, which makes the pathogen thrive. Most studies have a longer storage time and favourable environmental conditions associated with a higher aflatoxin accumulation [38][39][40].
The non-detection of AFB 2 in ICGV-03401 further boldens the resistance of this genotype, which could probably be due to the expression of genes that interfere with the aflatoxin biosynthetic pathway. This could mean apart from the concentration of specified aflatoxins, its absence could account for the resistance in a given genotype. Additionally, scenarios where non-aflatoxigenic A. flavus infection did not lead to aflatoxin production could be attributed to the presence of deleterious genes in the fungi that block the aflatoxin biosynthetic pathway. It is also possible that one or more aflatoxin biosynthetic genes may be absent, as reported by Commey et al. [37] when aflatoxin biosynthetic genes were compared between a non-aflatoxigenic A. flavus and aflatoxigenic A. flavus isolates. Non aflatoxigenic isolates have been used as biocontrol agents to reduce aflatoxin contamination in peanuts by 70%-100% in Ghana [41]. Similarly, Xu et al. [42] used non-aflatoxigenic strains to reduce aflatoxin B 1 accumulation in peanut kernels by 90%. In countries such as USA, Senegal, Nigeria, and Gambia, non-aflatoxigenic A. flavus are now serving as biocontrol agents to reduce aflatoxin production commercial in peanut production [43,44].

Conclusions
In conclusion, this study isolated and categorised A. flavus isolates into aflatoxinproducing isolates and non-aflatoxin-producing isolates from infected peanut genotypes using cultural techniques. Additionally, there were variations in the infection and colonisation of resistant and the susceptible peanut genotypes when inoculated with the aflatoxigenic and non-aflatoxigenic strains. The HPLC results confirmed that inoculations with the aflatoxigenic isolate resulted in the production and accumulation of aflatoxin B 1 , B 2 , and G 1 , whereas inoculation with non-aflatoxigenic A. flavus did not produce any aflatoxin. The most resistant genotype lacked one of the detected aflatoxins under HPLC. Therefore, aflatoxigenic and non-aflatoxigenic strains all have equal ability to infect peanut seeds, but peanut genotypes demonstrated varied resistance to these isolates and varied aflatoxin accumulation. IVSC technology can be used as a quick method to screen for resistance to A. flavus infection and aflatoxin accumulation. The peanut genotypes ICGV-03401 and L027B could serve as sources of donors for resistance to A. flavus infection and aflatoxin accumulation. Future studies could also be carried out on the utility of non-aflatoxigenic isolates as a potential source of biocontrol agents.

Study Area
This experiment was carried out at the Plant pathology laboratory of the CSIR-Savanna Agricultural Research Institute, Nyankpala, Ghana.

Preparation of Potato Dextrose Agar (PDA)
Culturing of A. flavus was carried out using Potato Dextrose Agar (PDA) medium (Oxoid) according to the manufacturer's procedures. The prepared PDA was amended with chloramphenicol to inhibit the growth of bacteria and then dispensed into the sterilised disposable Petri dishes to solidify in an antiseptic environment [45].

Preparation of Yeast Extract Sucrose Agar (YESA)
A total amount of 40.50 g of Yeast extract sucrose agar made up of yeast extract of 4.0 g, sucrose of 20 g, KH 2 PO 4 of 1.0 g, MgSO 4 of 0.5 g, and agar of 15 g was used and prepared according to the manufacture's procedures. It was amended with chloramphenicol to inhibit the growth of bacteria and beta-Cyclodextrin an aflatoxin inducing chemical respectively. This was dispensed into the sterilised Petri dishes in an antiseptic environment to solidify. Plates were exposed to Ultraviolet (UV) light to check for their fluorescence.

Source of Infected Peanuts for A. flavus Isolation
Infected peanut samples were obtained from a peanut processing centre at Nyankpala, Northern Region of Ghana. The infected seeds were collected and well packaged into a well labelled brown paper bag, and kept at room temperature until the time of isolation of A. flavus.

Isolation of A. flavus Isolates
With a modified isolation method by ref. [46], infected peanut seeds obtained were surface sterilised with 0.5% sodium hypochlorite solution for 1 min, rinsed in distilled water three times, and dried on tissue paper. Using sterilised forceps in the lamina flow hood cabinet, the surface-sterilised and dried seeds were placed onto the solidified PDA growth medium in the Petri dishes (10 seeds per plate) and incubated for 72 h at 27 • C.
The plates were visually observed and any visible mycelium on the seeds characterised by green-yellowish colouration was considered as the initial isolation criterion for A. flavus.

Obtaining Pure Cultures of A. flavus
Pure culture isolates were obtained by transferring mycelium with green-yellowish colour with sterilised inoculation needle from the previous plates and inoculating onto the centre of a fresh petri dish with PDA medium under aseptic environment in the lamina flow and incubating the plates for 10 days at 25 • C [46].

Identification of A. flavus
A. flavus was identified by observing morphological structures such as colony growth, conidia, texture, and colour [34,47] of the isolates obtained. Exposure of Isolates on PDA to Ammonia Vapour In 9 cm glass Petri plates, each isolate of A. flavus was inoculated in the centre of a solidified PDA medium and cultured for 7 to 8 days at 25 • C. The plates were subjected to 2 mL of concentrated Ammonia solution for 4 to 5 min and observed for change in colour of the under-side of colonies of aflatoxin-producing isolates. Those plates that changed colour from green-yellowish colour to orange-yellow pigmentation were classified as aflatoxigenic and those that did not change colour were noted as non-aflatoxigenic [48].

Radial Growth of Aflatoxigenic and Non-Aflatoxigenic Isolate on PDA
Four replicates of each isolate were plated on PDA and observed for their radial growth until the isolate with the fastest growth covers the entire plate. The area under the disease progress curve (AUDPC) was computed for each isolate growing on the PDA using the radial growth results for days 2, 4, 6, and 8 using the formula as described by Shaner and Finney (1977).
where n is the total number of observations, y i is an assessment of a disease at the ith observation, and t i is time at the ith observation.

Peanut Genotypes
Peanut genotypes less than 2 months old after harvest and free from any fungal contamination were used in this study. The three peanut genotypes, namely, ICGV-03401, L027B, and Manipinta, used in this study were obtained from the CSIR-Savanna Agricultural Research Institute, Nyankpala, Ghana. The ICGV-03401 have been reported to have resistance to A. flavus infection in vitro and similarly L027B have also been reported to have resistance to A. flavus infection in vitro (unpublished data). Manipinta is known to be susceptible to A. flavus infection in vitro.

Experimental Design
The experiment was laid out in a Completely Randomised Design (CRD) with three replications. The treatments were the three peanut genotypes and these were inoculated first with aflatoxigenic A. flavus in one experiment and in another experiment inoculated with non-aflatoxigenic A. flavus. Each plate served as a replicate with ten healthy peanut seeds.

Seed Sterilisation and Inoculation with Aflatoxigenic and Non-Aflatoxigenic Isolate
Preceding the inoculations, 30 peanut seeds with intact seed coats of each genotype were surface sterilised with 0.5% sodium hypochlorite and rinsed properly in sterile distilled water three times. The sterilised peanut seeds were placed in Petri dishes lined with moist Whatman No. 2 filter paper. Conidial suspensions were prepared from a 10-day-old culture of A. flavus isolate grown on PDA medium. Each seed was inoculated using 60 µL of suspension of A. flavus containing 1 × 10 6 spores per mL. They were incubated at room temperature for eight days [20,36].
After 8 days, the seeds were visually observed for seed infection by A. flavus by recording the percentage of seeds infested, which was shown by the presence of sporulating surface growth. Inoculations were performed using aflatoxigenic A. flavus and non-aflatoxigenic A. flavus.
Pathogen severity = Sum of pathogen rating Highest rating × Total number of kernel rated × 100

Detection of Aflatoxins Using High Performance Liquid Chromatography Sample Extraction
Aflatoxin was extracted using methods described by ref. [49] with slight modifications of using acetonitrile: acetic acid v/v (9:1) as the extraction solution. Using a Preethi Mixer Grinder (Tamil Nadu, India), peanut samples were homogenised. A weight of 2 g of the homogenised sample was quantitatively transferred into a 15 mL centrifuge tube, 5 mL of distilled water was added, and the tube was vortexed for 1 min. After allowing the sample solution to stand for 5 min, 5 mL of the extraction solution was added. Using the Genie Vortex machine (New York, NY, USA), the resultant mixture was vortexed for 3 min. Next, 1.32 g of anhydrous MgSO 4 and 0.2 g of NaCl were added to the mixture and vortexed for 1 min. This was followed by centrifuging the tubes for 5 min at 3300× g. The upper organic layer was then filtered through a 0.45 µm nylon syringe prior to injection. Finally, 50 µL of the filtered extract was injected into the high-performance liquid chromatographer (HPLC).

HPLC Analysis Technique
HPLC analysis was carried out based on AOAC Official Method 2005.08 (AOAC,2006) with Photochemical Reactor for Enhanced Detection (PHRED) for post-column derivatisation. A Cecil-Adept Binary Pump HPLC (Cambridge, UK) was joined with Shimadzu 10AxL fluorescence detector (Shimadzu Corporation, Tokyo, Japan) (Ex: 360 nm, Em: 440 nm) with Sunfire ® C18 Column (150 × 4.60 mm, 5 um). The mobile phase used was methanol: water (40:60, v/v) at a flow rate of 1 mL/min with column temperature maintained at 40 • C. LCTech UVE (Dorfen, Germany) was used for post-colum photochemical derivatisation. The aflatoxin mix (G 1 , G 2 , B 1 , B 2 ) standards (ng/g) of 5.02 ng/µL in acetonitrile from Romer Labs ® was used for matrix-based calibration. Aflatoxins in the samples were detected by using the retentions of the standard solution run and quantification was conducted using the calibration of curves of each respective toxin. Quality assurance for established by checking for precision and trueness by spiking blank samples with aflatoxins standard (Table 3). Blank samples were run periodically confined to the absence of aflatoxins. The coefficient of variation obtained for replicates was less than 15%.

Data Analysis
Data were subjected to analysis of variance using GenStat (12th Edition) version 12.0.0.3033. Treatment means were separated using the Least Significance Difference (LSD) at 5% significant level.