Arbuscular Mycorrhizal Fungi Induced the Content of Isoflavonoid that
tomato and carrot (Talavera et al., 2001), soybean (Morandi et al., 1984), olive (Castilo et al., 2006) grown on soil inoculated with nematodes have been showed. However, there was no information available about the role of AMF in protecting potato plants against root-knot nematodes, especially in Indonesia. Furthermore, the mechanisms in which AMF reduced nematodes population have not been cleared. Physiological changes in AM roots could change resistance to nematodes by increased production of inhibitory substance.
Plant that received nematode alone and mycorrhiza plus nematode recorded differential biochemical contents of proteins, total phenols and IAA (Nagesh and Reddy, 2004). Singh et al. (1990) showed that pre-occupation of the roots with AMF coupled with biochemical changes such as increases in lignins and phenols make Pusa Ruby resistant to root-knot nematodes. It seems that there is no single response in the interaction mycorrhiza-nematode and that the response depends on various factors, such as mycorrhiza-nematode, mycorrhiza species, plant species or environmental factors (Talavera et al., 2001).
The objectives of this study were: (i) to determine the effect of AMF on population of G.
rostochiensis on potato plant-nematode system; and (ii) to determine whether the reduction of nematode populations was associated with the production of inhibitory substance in plants inoculated by AMF.
Materials and Methods
A pot experiment was undertaken in a glasshouse of Faculty of Agriculture Universitas Padjadjaran from August to October 2010. Certified Potato (Solanum tuberosum L.) cv.
Granola seeds were planted in plastic pots (25 cm top diam.; 20 cm bottom diam.; 25 cm depth), filled with 5 kg of a sterile soil (soil analysis presented at Table 1).
Table 1. Analysis of Soil from Pangalengan West Java used in Experiment (Collected in July 2010)
Parameter Methods Value
pH H2O Potensiometri 6.5
pH KCl Potensiometri 6.0
C-org (%) Walkley & Black 5.52
N-org (%) Kjeldahl 0.49
P2O5 (mg/100g) HCl 25 % 11.86
K2O (mg/100g) HCl 25 % 17.91
CEC (cmol (+)/kg) Perkolation/NH4 Acetat pH 7 25.54
The treatment were number of spores of AMF per pot (0, 50, 100, 150) and juvenils of G.
rostochiensis (0 or 4000 per pot). Inocula AMF (Glomus sp.) consisted of spores were obtained from AMF pot cultures using Sorghum as host plants. The spores of AMF were applied according to the treatments one week before planting to enable spores to germinate.
Inocula of PCN G. rostochiensis for experiments were obtained by extracting eggs from soil at the potato plantation at Pangalengan West Java using methods described by Bridge and Starr (2007). The eggs were hatched and submersed in water for three days to obtain second-stage juveniles (J2). The nematodes treatment were applied one week after planting. Each treatment was replicated three times and pots were arranged on a glasshouse bench in a randomized-block design. Plants were watered daily to field capacity
and fertilized with 25-5-20 liquid fertilizer (50ml of 1/1,000 diluted Hyponex) at the time of planting. The experiment ended seven weeks after inoculation with the nematodes.
Seven-week-old potato plants were harvested . Potatoes were pulled from the soil and weight of shoot and roots of were recorded. The soil from each pot was sieved through a 2.5 mm mesh to separate the roots from the soil. Roots were washed free of soil, finely chopped, mixed thoroughly again with the soils, and stored in plastic bags. The nematode population in soil and roots (number of female eggs and juvenils) were assessed from soil samples by methods described by Bridge and Starr (2007). The percentage of the root system colonized by the mycorrhiza was assessed in subsamples of 0.5 g of roots at harvest time by the methods of Brundrett et al. (1996).
The production of inhibitory substance including isoflavonoid, Indole Acetic Acid and Chitinase were assessed from root samples by High Performance Liquid Chromatography (HPLC) at Laboratory of Research Center for Biology. Data were analyzed by two-way ANOVA using SPSS 16 program. When F values were significant, means were compared by the LSD test ( p_0.05).
Results Population of Potato Cysts Nematodes G. rostochiensis
Numbers of cysts of G. rostochiensis 100 g of soil was significantly lower in mycorrhizal plants than non-mycorrhizal plants . The number of cysts decreased as the number of AMF spores increased from 50, 100, and 150 per pot. The average reductions were 42, 60 and 86 % respectively (Table 2). The number of female was lower compared to control only when the AMF spores applied at the highest rate, with the reduction of 70%.
Table 2. Population of cysts and female of G. rostochiensis on roots of potatoes inoculated by spores of AMF
AMF spores per pot Cysts per 100 g of soil Female per g of roots
0 16,7 c 6,7 b
50 9,7 b 5,3 b
100 6,7 ab 5,0 b
150 2,3 a 2,0 a
Within each column, values followed by the same letter are not different (p>0.05) Mycorrhiza Colonisation and Content of Inhibitory Substance on Roots
The percentage of roots colonised by mycorrhiza increased according to the rate of number of AMF spores. However, no significant differences were observed in the percentage of roots colonized by the mycorrhiza in plants infested by PCN G. rostochiensis (Table 3).
Inoculation of G. rostochiensis significantly decreased the content of isoflavonoid in roots of potatoes up to 25% (Table 3). Increasing the spores number of AMF from 50, 100 and 150 per pot significantly increased the content of isoflavonoid 34%, 48%, and 64% respectively.
No significant differences were observed in the content of chitinase and IAA as the effect of AMF and PCN inoculation.
Table 3. Root colonisation by AMF (%), Content of isoflavonoid, chitinase, and IAA on roots of potatoes inoculated by AMF and PCN G. rostochiensis
Treatments AMF Root
Colonisation (%)
Isoflavonoid (ppm)
Chitinase (µg/g/hour)
IAA (ppm) AMF
(spores/pot)
0 0 9.8 a 0.13 a 13.6 ab
50 31 a 14.8 ab 0.13 a 10.3 a
100 37 a 18.8 b 0.13 a 16.3 b
150 52 b 26.9 c 0.14 a 13.5 ab
PCN (J2/pot)
0 36 a 22.9 b 0.13 a 13.9 a
4000 43 a 17.4 a 0.13 a 13.0 a
Within each column, values followed by the same letter are not different (p>0.05)
Discussion
Establishment of AMF in plant root systems is thought to contribute to biological control of plant diseases primarily by menas of stress reduction. The mechanismss including improvement of P absorption by plants; competition with the pathogen for infection sites and nutrients; morphological changes in roots and root tissues; micorbial changes in the mycorrhizosphere; and changes in chemical constituents of plant tissues (Suresh et al., 1985; Linderman, 1996). The use of AMF to protect plants against PCN G. rostochiensis could be a useful for the integrated management of root-knot nematodes in potato production (de Ruijter and Haverkort, 1999).
In general, this experiment showed that inoculation of AMF on potato plant-PCN system, reduced the number of female and cysts of G. rostochiensis. This is concured Sikora (1981) who found that the population of a Globodera rostochiensis Wollenweber reduced when potato plants inoculated by G. fasciculatum. Moreover, Suresh et al. (1985) stated that mycorrhiza reduced the number of nematodes larvae up to 50% and female nematodes were significantly lower in mycorrhizal plants. Ingham (1988) was also found that population of juvenils 75% lower when cofee, wheat and tomato plants were inoculated by Glomus mossea. Furthermore, Smith et al. (2001) confirmed that population of nematodes M. incognita on cotton plants reduced if the the level of mycorrhiza colonisation was higher than 50%. In this experiment, addition of 150 spore per pot resulted 52% root colonisation and highest reduction in nematode populations. The development of nematodes would inhibited when mycorrhiza penetrated the roots (MacGuidwin et al.,1985; Suresh et al., 1985).
In this experiment, the mechanisms on the reduction of PCN by FMA were likely because of direct competition for infection sites and production of isoflavonoid as one of inhibitory substance to nematodes (Table 3). Morandi et al. (1984) found that the concentration of isoflavonoid (phytoalexin-like) increased in mycorrhizal soybean plants and considered that isoflavonoid have a function as nematicide. Suresh and Bagyaraj (1985) assumed that the occurence of nematicidal substance in mycorrhizal roots was because of improvement of plant vigor and nutritions.
The biochemical defense response to mycorrhizal colonization against root-knot nematodes has been documented (Smant et al., 1997). Assay on the activities IAA oxidase enzymes in treated and healthy roots of tomato, indicated that IAA oxidase activities were maximum in G. fasciculatum colonized roots followed by the roots that received mycorrhiza plus nematode (Nagesh and Reddy, 2004). Demonstration of other change in chemical constituents due to AMF inoculation showed by Dehne et al. (1982) where concentration of anti-fungal chitinase were increased in AMF roots. However, this experiment did not show the difference in IAA nor Chitinase content as affected by AMF inoculation (Table 3).
The effect of AMF on growth of plant infected by nematodes were influenced by some factors including plant cultivar, species of nematodes and fungi, status of soil nutrients, time of inoculation and harvest.
The growth of mycorrhizal plants infested by nematodes were generally had smaller leaves and unhealthy (data not shown). AM fungi compensate some parts of the plants that demolished by nematodes. Ingham (1988) showed that the growth of mycorrhizal plants received nematodes were bigger than non-mycorrhizal plants. AM fungi would not colonise the site infected by nematodes and vice versa, the nematodes do not infect the site colonised by AMF.
In general, plant-parasitic nematodes and AM fungi commonly occur together in the roots or rhizosphere of the same plant, each having a characteristic but opposite effect on plant vigor. The obligately symbiotic AM fungi may stimulate plant growth, whereas the obligate plant-parasitic nematodes usually suppress plant growth. This experiment indicates that AM fungi have potential as biocontrol agents when both groups of microorganisms occur simultaneously in the roots or rhizosphere of the same plant.
Conclusions
1. Inoculation of spores of AMF at the highest rate (150 spores per pot) reduced the population in term of number of female and cysts of G. rostochiensis on potato plant-nematode system.
2. The reduction of nematode populations was associated with the production of inhibitory substance isoflavonoid in plants inoculated by AMF.
Acknowledgement
Research was supported in part by Hibah Bersaing Grant (2010) from Directorate General of Higher Education of Indonesia (Dikti).
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