BIOCHEMICAL CONTENTS OF PEPPER SEEDLINGS INOCULATED WITH PHYTOPHTHORA INFESTANS AND ARBUSCULAR MYCORRHIZA

The effect of interactions between Arbuscular Glomus etunicatum and fungus Phytophthora infestans on biochemical contents of pepper plants was investigated in a greenhouse experiment. The sugar contents (i.e. Glucose, fructose and sucrose) were higher in the control and mycorrhizal inoculated pepper seedlings and the lowest in pathogen inoculated seedlings. Free amino acids were the highest in the simultaneously inoculated pepper seedlings, while total phenol was found to be the highest in pepper seedlings inoculated with P. infestans. The levels of nitrogen, phosphorus and potassium varied in the inoculated pepper seedlings without any significant difference in the treatment. The results obtained suggest protective influence of mycorrhiza by enhancing the nutritional status of the inoculated pepper seedlings.


I n t r o d u c t i o n
Plant responds to mycorrhizal invections in a mutual symbiotic way, whereby the two organisms (Plant and mycorrhizal fungus) gain from each other.At the energy stage of the mycorrhizal infections to plant there is usually reduction in plant growth, which can result in Chlorotic symptom.This could be attributed to shift in the balance of establishment of mycorrhizal fungus and the host.Gains from the symbiosis occur through in the improved nutrition, increase in above ground biomass, and the ability of the plant to suppress or wipe out diseases completely is considered the usual benefit of mycorrhizal infection (Thompson, 1994).The positive impact of mycorrhizal infection on plant productivity via improved plant nutrition had also been extensively reviewed by Chanwaly et al. (1991).
On the other hand, the effect of pathogen infection on nutrient contents of plant products has been destructive or adverse to nutritional quality or plant.The loss in nutritional quality makes plant susceptible to infection.The complete loss of sugars in banana, carrot, tomato, water melon and other fruits or root tubers due to fungal pathogenic infestation has been reported (Odebode and Sanusi 1996; Odebode and Nwachukwu 1997; Oladiran and Iwu, 1992).In some instances there is a breakdown of the proteins to amino-acids in the host cells during infection, which leads to the increase in nitrogen content and decrease in protein content (Odebode and Sanusi, 1996;Arinze et al. 1976;Ogundana et al. 1970).
This paper reports the effects of AM mycorrhizal and a pathogenic fungus on nutritional content of pepper seedlings.

Plant materials and inoculation
Pepper seeds used in this study were obtained locally from a market in Ibadan, Nigeria.Seedlings of the plant were raised in trays (35x15x10 cm) filled with loamy tom soil sterilized at 121 o C at 10kg/cm 2 for one hour.Seedlings were transplanted after 2 weeks into plastic pots (20 cm diameter and 30 cm deep) filled with sterilized soil.
Phytophthora infestans was isolated from infected peppers in a local farm using baiting technique of Satisty and Hedge (1988).Pure culture of isolates were obtained by consequent subculturing on acidity potato dextrose agar (APDA).The pathogen inoculum was prepared as spore suspension obtained from 7 day-old cultures.The appropriate concentration was measured by using Hawskley heamocytometer.
The AM inoculum of Glomus etunicatum obtained from Soil Biology unit of the Department of Botany and Microbiology of the University of Ibadan was multiplied as pot cultures with Zea mays (L) and consisted of mixtures of soil and root fragments.The inoculation of seedlings with P. Infestans containing 4.0x10 5  spores was done for 30 minutes.Mycorrhizal inoculation was also introduced by weighing 30 grams of AM inoculum directly below each seedling and then drenched and covered back with the sterile soil in the plastic pot.Five treatments were used in this study.(i) Uninoculated control, (ii) Inoculated with P. Infestans, (iii) Inoculated with G etunicatum (iv) Inoculated with P. Infestans and G. etunicatum simultaneously and (v) At 2 weeks after inoculating pepper seedlings with the different single inoculations (i.e.pathogen and mycorrhizal separately) concomitant i.e. dual inoculation was carried out.These seedlings were left after inoculation in the greenhouse to grow and the biochemical activities were carried out at 2 weeks after inoculation.Each treatment was replicated three times and the planting was carried out in the greenhouse.The plant parts used for extractions were leaves and shoot.

Extraction of total phenol
The method of extraction used was a modified method of Odebode (1995).Five milliliter of each of the treatment was pippetted into 500 ml conical flask 50 ml, chloroform was added and shaken to extract for 2 hours before filtrating.The filtrate was retained for total phenol determination.

Determination of total phenol
The determination of total phenol was carried out as follows: 20 ml of the filtrate prepared above was measured into 100 ml conical flask; 2 ml of concentrated acetic acid was added with gentle warming.It was allowed to cool before adding 10 drops of dilute H 2 SO 4 (1:1) to the mixture; it was thoroughly shaken before adding 5 drops of 10% NaNO 3 and was allowed to stand for 45 minutes after mixing.At the end of the time interval, the reaction mixture was washed with 20 ml alcoholic ammonium hydroxide (4:6) solution into a 100 ml conical flask.It was cooled in ice broth and allowed to stand for 1 hour.The greenish-yellow mixture was filtered and read at 420 nm to determine the absorbence on a Cecil (CE☺595 Double Beam Digital U.V. spectrophotometar).

Extraction of sugar
One gram of oven-dried leaves and shoot were used following the method of Odunfa and Adey ele (1987).

Quantitative determination of sugar
This was carried out using the phenol sulphuric acid method of Dubuios et al. (1956).The optical density (OD) of the sugar was read on a Cecil (CE) 505 Double Ocean Digital U.V. spectrometar at 490 mm.

Extraction of free amino-acids
Dried leaves samples were extracted following the method of Oy eyiola (1990).

Quantitative determination of free amino-acids
Amino acids were determined quantitatively by measuring 5 ml of 0.219 citrate buffer into 0.5 ml of the extract from the treatment samples, 0.2 ml of 5% (W/V) ninhjydrin in methyl cellosolve and 1 ml of potassium cyanide methyl cellosolve (prepared by 0.IM potassium cyanide in 250 ml methyl (cellosove) were added and the resultant mixture was heated for 15 minutes at 120 o C. It was cooled under the running tap water for 5 minutes and brought to final volume with 5.7 ml of 60% isopropyl alcohol.The optical density (OD) was read at both 570nm and 440nm on a Cecil (CE) 505 double digital U.V. spectrophotometer.

Determination of nitrogen, phosphorous and potassium
On gramme dried leaves, a sample of each of the treatments, was used in the determination of nitrogen, phosphorous and potassium content of the pepper seedlings following the A.O.A.C. (1980) methods.

Results and Discussion
Pepper seedlings inoculated with pathogen alone had severe disease symptoms, while those inoculated with mycorrhizal alone had highest flowering and fruiting values.But pepper seedlings simultaneously or dually inoculated with mycorrhizal and pathogen also had good growth parameters especially where mycorrhizal was first inoculated before pathogen.The effects of the interactions on the biochemical contents of the pepper seedlings were shown in (table 1).The highest level of glucose, (2.50, 2.88, 5.95, 5.84) ug/ml fructose, and sucrose were found in the control and pepper seedlings inoculated with mycorrhizal alone, followed by pepper seedlings with simultaneous inoculation.Lowest sugar content of (2.0 ug/ml) was found in pepper seedlings inoculated with the pathogen.Total phenol and nitrogen were found to be highest in pepper seedlings inoculated with pathogen alone.Phosphorus was highest in mycorrhizal alone inoculated pepper seedlings.The statistical analysis showed that the levels of biochemical contents were not significantly different at (P<0.05) according to Duncan.The free amino acid contents were lower in all the treatments, also the proline.The total phenol even thought low, the level was nearly double in seedling inoculated with pathogen alone than that of control and mycorrhizal inoculated pepper seedlings.The results were the average of three replicates.
The initial increase in the level of sugars and free amino acids in the seedlings inoculated with mycorrhizal fungus alone indicated a protective mechanism of the pepper, while the increase in sugar also served as a source of energy for the pepper plants and thus increased its defense mechanism against pathogenic infection and also helped pepper seedlings in the symbiotic association with Mycorrhizal fungus.
The high level of total phenol in pathogen inoculated pepper seedlings alone indicated in-built resistance against pathogenic infection by the phenolic compounds, and also shows the presence of enemy (Williams, 1962).Postinfectional changes in the phenolic compounds of host plant is characteristic of host pathogen interaction in diseased plant ( Values with the same letter are not significatntly different according to Duncan's multiple range test (P<0.05).
On the other hand, the low level of total phenol in mycorrhizal inoculated plant may be a passive protective effect of mycorrhizal in that the plant does not have to build up many chemical defense mechanisms, an active defense mechanism as we have seen in pathogenic infections.Secondly, the decrease in the total phenol in mycorrhizal plants may also indicate a change in their physiology, both physically and chemically.Vandermolen et al. 1983;Osonubi et al. 1990 made a similar report that due to mycorrhizal infection into plants there is a physiological change that enables platns to resist infection by pathogen.Although the level of biochemical contents in mycorrhizal alone or simultaneously (pathogen and mycorrhizal) inoculated pepper seedlings may not be significant statistically.The increase in the level of biochemical contents showed healthy or balanced physiological stability of the pepper seedlings against pathogenic activity of phytophthora infestans, suggesting a protective influence of mycorrhizal via a strong nutrient availability.

C o n c l u s i o n
According to the obtained results of this investigation, it can be concluded that: Biochemical content of pepper seedlings inoculated with Ph. infestans and mycorrhizal fungi are undergoing changes.
The sugar contents of pepper seedlings were higher in the control and mycorrhizal inoculated than in pathogenic fungi inoculated seedlings.
Free amino acids are the highest in pepper seedlings simultaneously inolculated with mycorrhizal and patogenic fungi.
The nitrogen, posphorus and potassium level varied in the inoculated pepper seedlings without any significant difference in the treatment.
Our investigation suggests protective influence of mycorrhizal fungi by enhancing the nutritional status of inoculated pepper seedlings.