Aflatoxin in peanut is a major problem in worldwide and development aflatoxin resistant cultivars is required. The work presented in this thesis was conducted with the specific objectives to (a) develop a HPLC technique to quantify aflatoxins in peanut, (b) understand the variation in aflatoxin production as influenced by reproductive stage (R-stage), growing environment and genotype and (c) identify physiological and biochemical factors that are responsible for the genotype’s resistance or susceptibility of the production of aflatoxin.
To extract and quantify aflatoxins in peanut kernel, a rapid and cost effective technique has been developed using a UV-detection based HPLC system. The minimum detection limit of the current method is 0. 6 to 1ppb. This method was validated against two present techniques.
The experimental program was as follows. During 2001-02 three field experiments were conducted to examine genotypic variation at preharvest period. In experiment 1, two peanut genotypes were tested under mild water stress (MWS) and severe water stress (SWS) conditions. In experiment 2 and 3, ten genotypes were examined under drought conditions at two locations. In these experiments aflatoxins, RWC, pod leakage (%), pod water activity (aw). pod moisture content, sugars (glucose, fructose and sucrose) and amino acids (asparagine, trans-4-hydroxy methyl proline (MHP) and proline) were studied. Using the same ten genotypes, in experiments 4 and 5 during 2003-04, leakage of aflatoxins and aflatoxins in shells were examined. Under controlled environmental conditions (in vitro) seven genotypes that were grown under drought conditions from experiment 4 were screened for aflatoxin production.
The total aflatoxin was higher in SWS than MWS conditions but it decreased during pod filling period. In experiment 1, concentration of aflatoxins reduced from 69 to 6ppb and content from 2.3 to 1.i µg m-2 from 120 to 150 DAS. A similar pattern was also noted in experiment 2. This may be related to the fact that aflatoxins leaked out of pods and more of this occurred in mature and developing pods than juvenile pods. The decline in aflatoxins may also be related to the method of sample preparation. For analysing aflatoxins in R3-5 stage pods, shells were included with kernels, as shells were not possible to separate from kernels, whereas, in R6-8 only kernels were tested for aflatoxins. Shells were found to have higher aflatoxins compared to kernels.
The juvenile pods were more vulnerable for aflatoxin production than mature pods. A t any particular sampling date mature pods had more growth cracks but growth cracks were not apparently related to high fungal invasion. The higher pod moisture in younger pods than mature pods was associated with higher aflatoxins. The levels of sugars and amino acids were also higher in juvenile pods than in mature pods. Because of higher concentration of all sugars and amino acids in juvenile pods, they were more susceptible to Aspergillus invasion and aflatoxin production than mature pods.
There was peanut genotypic variation in aflatoxin levels in all experiments at different sampling dates. Among genotypes tested NC-7 produced higher levels of aflatoxin than Streeton in experiment 1 . Whereas in experiments 2 and 3, AX3-137 and AX2-92 produced higher aflatoxin than AX4-565. However, the coefficient of variation in these experiments was very high i.e. above 100% for total aflatoxin, suggesting there may have been large errors associated with sampling techniques and uncontrolled environmental factors, which confounded the observed genotypic differences.
Under in vitro conditions with reduced variability, genotypic differences were noted. The total aflatoxin level in peanut genotypes increased towards maturity. No genotype was completely resistant to aflatoxin production at unit substrate level. The genotypes AX4-565, Streeton and P4-3 produced higher level of total aflatoxin than other genotypes. However relationship of aflatoxins between field and in vitro studies was poor.
In conclusion, the present research established a simple and cost effective method to analyse aflatoxins. The identification of genotypic variation for aflatoxin production at field level was difficult due to the observed large sample variation in aflatoxin. Under water stress conditions due to high pod moisture content, sugars and amino acids the juvenile pods were more susceptible for aflatoxin contamination than mature pods. Data suggested that aflatoxins leaked into the soil during pre harvest period, and that the peanut shell appeared to have more aflatoxins than kernel, but these points need to be confirmed first.