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Aflatoxin concentrations in cv. TMV2 (Fig. 4.13a) rose more quickly and attained higher levels than cv. Jll (Fig. 4. 13b). By 72 hours, cv. TMV2 had attained 5.22 ppb aflatoxin, predominantly B, and Gi and at 216 hours reached 5803 ppb total aflatoxin (Fig. 4. 14a). In comparison, samples of cv. Jll contained 0.3 ± 0.01 ppb aflatoxin Bi by 72 hours and 168 ppb total aflatoxin by 168 hours (Fig. 4. 14b).

4. 3. 5. 3 Fungal growth

Paradoxically fungal growth occurred more quickly in the resistant cv. Jll, than in the susceptible cv. TMV2 (Table 4.3). Within 72 hours, glucosamine content of cv. Jll samples had reached 42.7 ±

1.3 pmoles/g. d. wt. , rose to 44.7 ± 0 . 7 pmoles/g. d. wt. by 96 hours and levelled off at 48.4 ± 1 . 2 pmoles/g. d. wt. by 120 hours

(a visual fungal growth rating of between 3 and 4).

Concentrations of glucosamine from samples of cv. TMV2 had reached 27.90 ± 1.3 pmoles/g. d. wt. by 72 hours (visual growth rating of 2) and rose steadily to 41.90 ± 1 . 6 pmoles/g. d. wt. by 216 hours after inoculation (Table 4.3).

Table 4.3,

MEASUREMENT OF FUNGAL GROWTH ON GROUNDNUT CULTIVARS TMV2 AND Jll INOCULATED WITH A. parasi ticus.

HOURS AFTER GLUCOSAMINE INOCULATION pmoles/g d. wt' J l l 72 42. 7 ± 1.3 96 44. 7 i 0,1 120 46. 4 ± 1.2 144 48. 7 ± 1. 1 165 48. 6 ± 0 .9 TMV2 72 27. 9 ± 1.3 120 33. 2 ± 2. 4 158 38. 9 + 1.5 216 41. 9 ± 1.6

• Results are the results of three replicates ± S. D.

4.5 DISCUSSION

For many years the aflatoxin problem in groundnut was linked more to the postharvest period than to the period of pod development in the soil. Hence, interest was focussed mainly on groundnut products such as meal and cake.

Resistance to A. flavus invasion and colonisation of rehydrated, stored, dried seeds has particular relevance when aflatoxin contamination is largely postharvest. However, significant aflatoxin contamination can occur before harvest

(Davidson et al. , 1983; Mehan et al. , 1986).

Recently there has been considerable research into the possibility of genetic resistance in groundnuts to seed infection and aflatoxin contamination in the field (Davidson et al.,1983; Mehan and McDonald, 1984a,b; Kisyombe et al. ,1985; Mehan et al. , 1986), Several groundnut lines with resistance to A, flavus and greater yield potential have been bred (Mixon 1986, Rao et al. , 1989). The nature of resistance appears complex: resistance mechanisms may operate at the pod surface, in the pod tissue, at the testa or within the cotyledons. In addition the maturity of these tissues must also be considered.

Maturity (or stage of development) influenced the type and amount of phytoalexins induced. Immature samples consisted of fused pod, testa and cotyledon tissue. The first line of defence was the fleshy pod tissue and this was found to exhibit a phytoalexin response when challenged (Fig. 4.5) by aflatoxigenic fungi. The phytoalexin response is absent from mature groundnut pod and testa

samples as the tissue is dead and lignified (see Chapter Three) providing a mechanical barrier for the cotyledons. The phytoalexin response of stage 7 and stage 8 fruit to inoculation by aflatoxigenic fungi depended on the isolate used. A. flavus IMI 91019bi, a white mutant strain with increased aflatoxin production

and A, parasiticus IMI 120920, a strain capable of producing all

four aflatoxins, were able to grow sparingly on stages 1 and 2, 4, and 6 of cv. TMV2. However, A. flavus IMI 93803, a non-toxic isolate, was unable to colonise any of the samples. All three isolates induced arachidin III and IV and a possible third phytoalexin arachidin I or II. Amounts varied according to tissue challenged, but, the most aggressive strain. A, flavus IMI 91019bi, always elicited the highest concentrations and the non- aflatoxigenic strain IMI 93803 the lowest.

These results suggest that contact between the parasite and cell wall of the groundnut tissue induced the accumulation of phytoalexins. Therefore, the more aggressive, invasive, isolates induced a greater phytoalexin response than the non-toxic isolate. Interaction between the fungus and groundnut tissue may be enhanced either by enzymes that are normally present within the host or by enzymes that are activated or synthesized in response to the infection process, eg. chitinase (Hadwiger and Loschke, 1981) and p-glucanases, which degrade the cell wall of the parasite, releasing elicitor fragments (Pegg and Young, 1981; Young and Pegg, 1982). Cell walls of Phytophthora megasperma contain a p-glucan fraction which was highly active in eliciting the accumulation of phytoalexins in soyabean (Ayers et al. , 1976).

Enzymes may have been secreted by the Aspergilli isolates, eg. pectinase, causing the release of elicitors from the cell wall of the groundnut tissue. These elicitors may have interacted with the nucleus of the groundnut cells initiating phytoalexin production. A highly purified glycoprotein from the culture filtrates of Rhizopus

stolonifer proved to have pectinolytic activity and to be an

elicitor of casbene synthetase activity in castor bean (Lee and West, 1961) and of pisatin in pea (Walker-Simmons et al,, 1984).

It is possible that the phytoalexin response was suppressed, in some instances, by compounds present in the cell wall of the parasite. Such material may have been specific to those races of

Aspergillus flavus/parasiticus, which suppressed the phytoalexin

response in groundnut tissue, facilitating the process of colonisation eg. inoculation of cv. TMV2 with A, parasiticus. The production of acetylenic phytoalexins by cultures of safflower

iCarthamus tinctorius) in response to an extract from Alternarla

carthami was suppressed by brefeldin A (100 pmol/dm), which is a

macrolide toxin produced by the fungus (Tietjen, 1964).

The rapid and high phytoalexin response of seed of cvs. Ah7223 and UF71513 to inoculation with A. parasiticus may have been responsible for retarding fungal growth. These cvs. were tested on receipt from India. This was not true for cvs. Jll and TMV2. After 9 months at 15"C the seed was inoculated with A. parasiticus.

Although the phytoalexin response was higher and aflatoxin production was slower and attained lower concentrations than susceptible cv. TMV2, fungal growth was greater. This suggests that Jll may inhibit aflatoxin synthesis. Damage may have occurred

during curing of the seed and this could have led in its worse form to dry, cracked testae and split cotyledons. As described in Chapter Three, storage may be responsible for the poor performance of this sample of cv, Jll; the phytoalexin response was certainly impaired in both cvs. after 9 months storage at 15*0 (see Fig, 3,2 Chapter Three).

Maturing groundnut samples (stage 7 development) of cv. TMV2 accumulated aflatoxin in response to A, parasiticus slowly and had low final concentrations (141 ppb). Resveratrol was not detected until 10 days after inoculation. TLC analysis revealed antifungal activity suggesting that phytoalexins are only part of the resistance process. Mature samples (stage 6) accumulated resveratrol by day 4 but aflatoxin concentrations were high (Fig. 4.6). Stages 1, 2, 4 and 6 accumulated arachidins IV and III and another compound, possibly arachidin I or II. Resveratrol may have been produced but it would not have been detected as the samples were extracted by facilitated diffusion (section 2.2),

Mature groundnut seed cv. TMV2 sent from ICRISAT and propagated at the University's greenhouse only accumulated resveratrol and several unknown compounds (Table 4.2 and Fig. 4.8). The same seed tested on receipt abiotically (Chapter Three Fig, 3.2) and 9 months later biotically (Fig. 4.11) accumulated resveratrol and arachidin III. Differences in phytoalexin content may have been due to the enviromental conditions experienced by the plant as it grew in the field, growth chamber or greenhouse. If the composition of the seed has been changed this may lead to differences in response to infection.

Cv, Jll is classfied resistant, used commercially in India, and described as highly desirable because of its resistance to aflatoxin production (Tulpule et al., 1977). Other workers have found that only one sample out of eight of cv. Jll collected from different agroecological regions of India showed resistance to A.

flavus (Mehan et al., 1986). This variation may have been due to

lack of uniformity in the seed used for sowing the crops at the eight different locations, or to climatic and edaphic factors affecting the chemical composition of the seed in different ways at the various locations.

LA « 4) W C « iO k o * 3 1.34917 + 2.3377e-2x R^2 = 0.997 2 1

0

0

Fig. 4.1. An example of a standard curve for glucosamine, concentration ranges from 0 - 100 pg/ml. Each point is the mean of three replicates.

Data for Fig, 4. 1. in Appendix 4, Table 4.4.

Fig. 4.2. Groundnut cv. TMV2 at two different stages of development: (a) stage 2, pericarp or pod was fused with the testa and appeared fleshy, cotyledons were small and white in colour; (b) stage 6, pod had started to lignify but was still fleshy, testa was pink in colour and fleshy, cotyledons were well developed.

IZO^TSO

h D A i S

Fig. 4. 3. Fruit of groundnut cv. TMV2 at stages 1 and 2 of development. Samples had been inoculated with either A. flavus IMI 91091bi or A, parasiticus IMI 120920 six days previously. Fungal growth was visible, rating 1, on samples inoculated with A, flavus.

a

Fig. 4.4. (a) Pod of groundnut fruit at stage 6 of development. The pods were washed^hree times with SDW and inoculated with h. flavus

IMI 93803. There was no sign of fungal proliferation after incubation for 6 days, (b) As in (a) but inoculation was at stage 4 of development.

G3 341 nm Q arachidin I V

0 a'achidin III

Fig. 4.5. Phytoalexin accumulation in groundnut fruit of cv. TM\'2 at different stages of development, inoculated with either A.

flavus IMI 93803 (a), A. parasiticus IMI 120920 (b) or A. flavus

IMI 91091bi (c). 1 = fruit at stage 1 & 2, 2 = fruit at stage 4, 3 = fruit at stage 5.

Data for Fig. 4.5. in Appendix 4, Table 4.5

10000 1000 100-^ l ü S t a g e 7 E 3 S t a g e 8 Days a f t e r inoculation

Fig. 4.5. Total aflatoxin content of cv. TMV2 infected with h.

parasiticus at stage 7 and stage 8.

Data for Fig. 4.6. in Appendix 4, Table 4.6.

10000 -à X o OB Œ 1000 /%// □ G2 1 0 0 - ^

Fig. 4. 7. Aflatoxin content of stage 7 and stage 6 seed of cv. TMV2 inoculated with A. parasiticus. 1, 2, 3 = stage 7 at 4, 6 and 10 days respectively after inoculation, la, 2a, 3a = stage 8 at 4, 8 and 10 days respectively after inoculation.

Data for Fig. 4.7. in Appendix 4, Table 4.7

-Ï- - '

Fig. 4.6 Antifungal activity of stage 7 and stage 6 TMV2 seed inoculated with A. parasiticus, a, b = stage 7 seed at 4 days after inoculation, c, d = stage 6 seed at 4 days after inoculation, e, f = stage 7 seed at 8 days after inoculation, g, h = stage 6 seed at 6 days after inoculation, i, j = stage 7 seed at 10 days after inoculation, k, 1 = stage 8 seed at 10 days after inoculation.

Data for Fig. 4.6. in results section of chapter. Table 4.2.

CCI «• © S 1 9 2 240

O arachidin

0 arachidin

[3 resveratrol

Fig. 4.9. Phytoalexin accumulation of Ah 7223 seed inoculated with

A. parasiticus over time.

Data for Fig. 4.9. in Appendix 4, Table 4.6.

E3 arachidin. !V