¹Department of Agronomy, Chiang Mai University, Chiang Mai, Thailand.
²Lampang Agricultural Research and Training Center, Lampang, Thailand.
³Department of Agriculture and Biological Engineering, University of Florida, Gainesville, FL, 32611-0570, U.S.A.

Abstracts

Media summary

Key words

Aspergillus flavus, Root exudates, Peanut genotypes, Hydroponic system, Water deficit

Introduction

Recent research conducted by Ingram et al. (1999) showed that a mini-rhizotron system could be used to observe A. flavus growth on peanut roots and pods in situ with peanut grown in containers and inoculated with a strain of A. flavus containing a green fluorescing protein (GFP) developed by Jeff Cary. Ingram et al. (1999) observed that A. flavus populations, as estimated by amount of fluorescence, increased at peanut root and pod surfaces, particularly under dry soil conditions. A. flavus populations appeared to be greater on roots and pods of drought-susceptible peanut genotypes than on roots and pods of drought-resistant genotypes. Root exudates, sloughed cortical cells and leachates may provide growth substrate to promote A. flavus population increase. Amounts of exudate are likely to differ among genotypes and have been shown to increase in response to stress in other plants. We hypothesize that root exudates may explain differences in A. flavus population development on root surfaces, and that such genetically-related differences may be exploited to increase aflatoxin resistance of peanut. The objectives of this experiment were to test this hypothesis by (i) estimating the amount of root exudates from roots of different peanut genotypes, (ii) quantifying the relationship between root exudates related to drought resistance, and then (iii) evaluating the effect of exudates on A. flavus populations in soil.

Material and methods

Four peanut genotypes (419CC: drought and aflatoxin susceptible; 511CC: drought and aflatoxin resistant; Tainan 9: commercial variety in Thailand; and Luhua 11: recorded as aflatoxin resistant variety from China) were pre-germinated between paper for 3-5 days and then seedlings were transplanted into the hydroponic system with half-Hoagland’s solution. Each container consisted of 4 plants (each treated as one replicate) of each genotype. The containers were fitted with an automated pumping system. At flowering stage, 30 days after planting (DAP), water-deficit stress was imposed on half of all peanut containers using poly-ethylene glycol 4000 (PEG) in deionized water (50 mg/ liter of nutrient solution). 24 hours after imposing stress, 4 plants of each peanut genotype were sampled and their root systems were transferred to containers of deionized water (500 ml) for 24 hours. Root exudates were collected from different genotypes in deionized water and stored at -20°C. Peanut plants were separated into plant shoot and root portions, roots system were stained with methyl violet in 95% of ethanol (2.5 g methyl violet/ 250 ml 95% ethanol and then diluted to 1 ml/ 100 ml H₂O). Half of the exudate solutions was applied in 5 ml aliquots using a hypodermic needle to sterile sand (20 g) mixed with the spore suspension of GAP and Kim (J. Cary) strains of GFP A. flavus (1 ml) on the petri dish. All petri dishes were incubated at room temperature (about 30°C) for 5 days and then growth of GFP A. flavus populations in the sand was estimated by the Petri plate dilution technique, using M3S1B medium (no added glucose) which was specific for A. flavus culture (Griffin and Garren 1974). Results were recorded as colony-forming units (CFU) of A. flavus group per gram sand. The other half of the root exudate solutions was analyzed for the amount of sugar (glucose, fructose and sucrose) by HPLC (mobile phase: Acetronitrile: H₂O (75:25); flow rate: 1.5 ml/minute; injection volume: 25 μl; sensitivity: 1.0:E-07 RIU/mV; HPLC column: packed column, 5 μm, length 250 mm). In addition, four peanut genotypes were grown with the sterile sand in the small containers. The control treatment was sterile sand without plants. All containers were irrigated with half-Hoagland’s solution. At 30 DAP, 20 g of sand from the root zone was sampled from all containers. This was mixed with 1 ml of spore suspension of A. flavus and incubated at 30 °C for 5 days. The population of A. flavus in the sand was estimated by the Petri plate dilution technique. Growth of A. flavus populations was recorded as colony-forming units of GFP A. flavus group fungi per gram sand. The stained root system was analyzed for total root length, root surface area, root volume and average root diameter using a flatbed scanner and WinRhizo software (V. 4.0 B, Regent Instrument Inc. 4040 Blain St., Quebec Q2B 5CB Canada.)

Results

The root exudates of four peanut genotypes were analyzed for glucose contents which showed no significant differences (Table 1). The mean of glucose content under non-stress condition could suggest that Tainan 9 genotype was greater in glucose content than 511CC, Luhua 11 and 419CC genotypes, respectively. Under stress condition (induced by PEG), 511CC and Luhua 11 genotypes may have released more glucose from root than the non-stress condition. The data in Table 1 also show that the fructose and sucrose contents of four peanut genotypes do not differ significantly and the stress condition had no effect on the release of fructose from root system. On the contrary, stress condition by PEG 4000 had an effect on the release of sucrose. Under stress condition, genotype 419CC tended to have greater sucrose content than other genotypes. Under non-stress condition, 511CC genotype did not release sucrose at all. Thus, water deficit in the peanut plant had been shown to increase the release of exudates from root, which may occur because of damaged root cell or injured cell membrane.

Table 1. The content of glucose, fructose and sucrose released from root system of four peanut genotypes under non- stress and stress condition.

Half of the stored root exudates solution was cultured with GFP A. flavus fungi. It was found that A. flavus fungi had high level of colonization but it occurred less than the stock of spore suspension (stock: 6.05 x 10⁶ spores/ ml). The spore suspension stock also had the active spore and non-active spore so the active spore could only germinate. The sugar substrates in exudate solutions might be less, so that the substrates were not enough to stimulate the A. flavus fungi growth (Table 2). The A. flavus colonization of each peanut genotype were significantly different and 419CC and Luhua 11 genotypes had highest level of A. flavus colonizing. These genotypes may have high level of sucrose and glucose for the substrates of A. flavus development. Under non-stress condition, Tainan 9 genotype exuded more glucose and fructose than sucrose, and may have no effect to stimulate the germination of A. flavus.

In the case of A. flavus cultured with the natural sand which had been grown to the peanut plants, it was found that 419CC, Luhau 11, Tainan 9 and 511CC genotypes were colonized in sand by A. flavus at 12.5 x 10⁴, 10.0 x 10⁴, 9.5 x 10⁴ and 9.25 x 10⁴ colonies per gram sand, respectively. The control treatment was colonized by A. flavus at 5.75 x 10⁴ colonies per gram sand. There were significant differences between sand on which the peanut plants were grown and were not grown. Root exudates (glucose, fructose, sucrose) are exuded from the injured and non-injured root system of peanut genotypes (Hale and Griffin 1975). Thus, the root exudates certainly had an effect to increase the A. flavus growth in sand.

Table 2. The colonization of Aspergillus flavus cultured with the root exudates solution of four peanut genotypes.

a b c = statistically significant at 5% level; mean ± standard error of mean

Total root length, root surface area, root volume and root average diameter were measured by using a flatbed scanner and WinRhizo software and showed no significant differences, except the total root length. Tainan 9 genotype was higher in root length than 511CC and Luhua 11 genotypes, respectively (Table 3). The amount of root exudate and total root length suggested that Tainan 9 and 419CC genotypes had larger root length and exuded more glucose, fructose and sucrose sugar than 511CC and Luhua 11 genotypes. So it might indicate that the large root length of peanut genotype could leak more exudates from the root cells under stress and non-stress condition.

Table 3. Total root length of four peanut genotypes measured by a flabted scanner and WinRhizo program.

a b c = statistically significant at 5% level

Conclusion

The study showed that stress condition promoted greater exudation of sucrose than glucose and fructose. There were no significant differences observed among peanut genotypes for sugar exudation. The drought-resistant peanut genotype (511CC) may have exuded less sucrose than 419CC genotype which is drought and aflatoxin susceptible, and no sucrose was found in the 511CC genotype under normal condition. There was a tendency for more sugar to be exuded from large root length peanut genotypes (419CC and Tainan 9) than from the small root length genotypes (511CC and Luhua 11). The results indicated that amounts of root exudate increase in response to stress for A. flavus population development under soil. This means that sucrose may support A. flavus colonization under stress condition.

References

Griffin, G. J., and K.H. Garren (1974). Population levels of Aspergillus flavus in Virginia peanut field soil. Phytopathology 64, 322 –325.

Hale, M. G., Griffin G. J.(1975). The effect of mechanical injury on exudation from immature and mature peanut fruits under axenic condition. Soil Boil. Biochem. 8, 225- 227.

Ingram, K. T., G. F. Patena, and C. C. Holbrook (1999). Drought and temperature effect on aflatoxin resistance peanut. Paper presented to 1999 Multicrop Aflatoxin Elimination Workshop. 20- 22 October, 1999, Atlanta, GA, USA.