Arbuscular Mycorrhizal Fungi Colonization in Upland Rice as Influenced by Agrochemical Application

Rice Science, 2009, 16(4): 307–313 Copyright © 2009, China National Rice Research Institute. Published by Elsevier BV. All rights reserved DOI: 10.1016/S1672-6308(08)60095-5

Arbuscular Mycorrhizal Fungi Colonization in Upland Rice as Influenced by Agrochemical Application Velu RAJESHKANNAN1, Chettipalayam Samiappan SUMATHI1, Sellamuthu MANIAN2 (1Rhizosphere Biology Laboratory, Department of Microbiology, Bharathidasan University, Tiruchirappalli 620 024, India; 2 Department of Botany, Bharathiar University, Coimbatore 641 046, India) Abstract: Mycorrhizal status of rice under upland conditions was studied using potted seedlings. Percentage of arbuscular mycorrhizal fungi (AMF) root colonization varied between 17.35% and 37.18% over an age series of 7 to 70 days old rice plants. AMF root colonization was increased up to 35–42 days, beyond which the root colonization steadily declined. The th

vesicles appeared after two weeks and reached their maximum intensity on the 35 day. The arbuscules were formed late on the 42

nd

th

day (2.93%) and slightly varied up to the 70 day (3.03%). Higher dosage of urea application suppressed plant

growth whereas the superphosphate treatment had no marked impact on plant growth. Generally, application of these agrochemicals registered less influence on the hyphal colonization of AMF in rice plants, whereas arbuscular colonization was adversely affected by higher dosages of fertilizers. There were pronounced decreases in both the plant growth and their AMF colonization due to the application of systemic fungicides, carbendazim and thiophanate methyl. The application of single sprays of fungicides was less deleterious over multiple sprays. Key words: Oryza sativa; arbuscular mycorrhizal fungi; agrochemical; upland rice

Rice is an important staple food for human society and it is grown during wet season under assured rainfall or irrigation in India. Arbuscular mycorrhizal fungi (AMF) form symbiotic association with varied plant species grown under agricultural cultivation areas. Reports on AMF association with rice plants are available both under upland [1] and lowland conditions [2]. AMF is directly and/or indirectly attributed to an improved nutrient status in land by forming association with plants. It increases the plant productivity by increasing the phosphate uptake [3]. Agrochemicals are essential inputs for boosting the outputs of various crops. Elaborate application of agrochemicals like fertilizers provides additional nutrients; pesticides and fungicides protects from the pest and fungal diseases. Some researchers have reported that the addition of P typically reduces the extent of AMF formation [4-6]. However, these studies have failed to distinguish the soil or the host-fungus interactions. Dhillion and Ampornpan [7] reported significant reduction of shoot and root growth of mycorrhizal plants as compared to non-mycorrhizal plants when the plants were supplemented with P and N, and suggested that the AMF association may be a carbon drain for the Received: 11 March 2009; Accepted: 7 July 2009 Corresponding author: Velu RAJESHKANNAN ([email protected])

young rice plants. It is therefore, agrochemicals affect the occurrence and distribution of AMF in several ways. The main objectives of this work are to observe the extent of AMF root colonization in upland rice and to assess the effect of certain fertilizers and systemic fungicides on the rice plant growth, biomass and development of AMF colonization.

MATERIALS AND METHODS Area description Rhizosphere soil samples (5 random samples) were extensively collected from a rice field of Thondamuthur (latitude 11°02ƍ N and longitude 76º58ƍ E, at the elevation of 409 m), Coimbatore, India. Soil substrate The soil had an initial pH of 7.9, 3.3 mS/cm electrical conductivity (measured using a bridge meter), 11.2 mg/kg nitrogen, 0.5 mg/kg phosphorus and 15.8 mg/kg potassium. The rice field soil of 2.0 kg per pot was filled in a 20 cm×12 cm plastic container. Cultivation of rice seedlings Rice seeds (Oryza sativa L. var. Ponni) were obtained from Tamil Nadu Agricultural University,

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Coimbatore, India. Healthy and uniform sized seeds were selected and soaked in water overnight and kept in shade for 12 h. The germinating seeds were spread on the surface of the soil in the plastic pots. The growing seedlings were maintained under wet conditions till the seedlings were harvested for the assessment of vesicular arbuscular mycorrhizal colonization. Agrochemical treatment Two commonly used nitrogen and phosphate fertilizers in rice fields viz., urea and superphosphate were applied to the rice seedling growing pots at three different dosages, i.e. 0.5, 1.0 and 2.0 g per pot containing 2.0 kg of rice field soil while the concentrations are referred as 0.25, 0.50 and 1.00 g/kg, respectively. The concentration of 0.50 g/kg refers to the recommended rate (100%) of field application while 0.25 and 1.00 g/kg refer to 50% and 200% of the recommended rate, respectively. Generally these fertilizers were applied over the surface of the soil and in order to assess the unique effect of fertilizers and fungicides on AMF colonization. The fertilizers were applied separately to the 10-day-old seedlings and observations were made on the 42nd day. The efficacy of two commonly used systemic fungicides differing in their active compounds viz., Bavistin (carbendazim) and Roko (thiophanate methyl) were tested by giving different number of spraying times at the concentration of 2 g active ingredient in l L water. After germination, single spray treatment was given on the 10th day, double spray treatments on the 10th and 20th days and the triple spray treatments on the 10th, 20th and 30th days. On the 42nd day, the growth and AMF root colonization of rice seedlings were observed. All the treatments were done in triplicates. Preparation of roots and AMF assessment The plants were harvested along with their entire root system at specified ages. The roots were thoroughly washed, cut into 1 cm bits and fixed in FAA. These root bits were cleared by boiling in 10% KOH for 10 min, then washed in water, acidified with 5 mol/L HCl for 5 min and stained with 0.05% tryphan blue in lacto phenol. The percentage of root colonization (percentage of hyphae + percentage of arbuscules + percentage of vesicles) was determined according to a magnified

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intersection method [8]. Estimation of soil nutrient content The total nitrogen (N) and available phosphorus (P) were determined respectively by the micro-Kjeldahl and the molybdenum blue methods [9]. Exchangeable K was extracted from the soil in ammonium acetate solution (pH 7.0) and measured using a digital flame photometer [9]. Statistical analysis Data were statistically analyzed by the Analysis of Variance (ANOVA-93 IRRISTAT, 1993) and means were separated using the Duncan’s Multiple Range Test. The data of AMF root colonization were analyzed by the arcsine-transformed analysis.

RESULTS Growth of rice seedlings and their mycorrhizal status The growth of rice seedlings under upland conditions is shown in Fig. 1. On the 7th day after germination, the shoot height was 6 cm, which increased gradually and reached 15 cm on the 70th day (Fig. 1-A) since it is a dwarf variety. Similarly, the mean root length of 5 cm was recorded on the 7th day and reached 11.8 cm on the 70th day. Shoot dry weight and root dry weight gradually increased based on the shoot height and root length, which varied between 20–40 and 16–27 mg/plant, respectively, over an age series of 7 to 70 days old plants (Fig. 1-B). But the ratio of root to shoot dry weight was reduced from 7 to 70 days old plants (Fig. 1-C). The extent of arbuscularmycorrhizal hyphal root colonization ranged between 17.35% and 37.18% in rice plants at different rice ages is presented in Fig. 2. The colonization of hyphal structures was evident even on the 7th day after germination. The hyphal colonization steadily increased from 17.35% and reached its maximum to 37.18% on the 42nd day. Thereafter, the percentage of root colonization of hyphal structures declined steadily to 28% on the 70th day. The vesicular structures were first observed in the 14-day-old seedlings at the intensity of 9.8%, which gradually increased to 17.9% on the 35th day. The vesicular colonization declined to

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Velu RAJESHKANNAN, et al. AMF Colonization in Upland Rice as Influenced by Agrochemical Application

Fig. 2. Extents of arbuscular mycorrhizal root colonization on different age rice seedlings under upland conditions.

Effects of fertilizers on the growth, biomass and mycorrhizal colonization

Fig. 1. Growth and biomass of different age rice seedlings under upland conditions.

8.3% on the 70th day and they appeared as globose to sub-globose as well as in chains. The arbuscular colonization was observed much later on the 42nd day at 2.9% and slightly varied up to the 70th day.

The application of urea and superphosphate has increased the growth of rice plants when compared to the control (Table 1). The recommended dosage (0.50 g/kg) of urea had positive and pronounced effects on the hypotrophy of rice plants when measured in terms of shoot height, root length, shoot dry weight, root dry weight and ratio of root to shoot dry weight. Whereas the higher dosage of urea (1.00 g/kg) suppressed the growth of rice plants compared to the recommended dosage as well as the dosage lower than the recommended. In the case of superphosphate treatment, the similar type of results was observed. The fertilizer effect in upland rice on the mycorrhizal root colonization status registered to have less influence and fluctuating results on the hyphal and vesicular colonization, whereas the arbuscular colonization was adversely influenced by the higher dosage of these fertilizers (Fig. 3). Urea at the rate of 0.25 g/kg was inhibitory to the arbuscular development.

Table. 1. Effects of fertilizers application on the growth and biomass of rice plants under upland conditions (assessed on the 42nd day after germination). Treatment

Dosage (g/kg)

Shoot height (cm)

Root length (cm)

Shoot dry weight (mg/plant)

Root dry weight (mg/plant)

Ratio of root to shoot dry weight

Urea

0.25 0.50 a 1.00 0.25 0.50 a 1.00

30.36 d ± 1.4 36.26 e ± 0.7 21.14 b ± 0.4 4.94 a ± 0.1 22.64 b ± 0.2 29.28 d ± 0.3 25.92 c ± 1.1

13.68 b ± 0.8 19.74 c ± 0.4 7.62 a ± 0.2 7.38 a ± 0.3 8.42 a ± 0.2 15.94 b ± 0.2 9.96 a ± 0.3

53.47 d ± 0.3 54.63 d ± 0.8 29.27 b ± 0.7 23.18 a ± 0.7 34.35 c ± 0.2 36.59 c ± 0.6 25.13 a ± 1.0

35.71 c ± 0.3 43.00 d ± 0.6 25.24 b ± 0.7 12.71 a ± 0.6 25.27 b ± 0.3 36.47 c ± 0.3 26.51 b ± 0.4

0.72 b ± 0.03 0.84 d ± 0.03 0.71 b ± 0.06 0.53 a ± 0.04 0.80 c ± 0.04 0.89 e ± 0.04 0.82 d ± 0.06

Control Superphosphate

a

Means followed by the common letter(s) are not significantly different at 5% level according to DMRT. Mean± SE at 1% level of significance. Recommended dosage.

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Fig. 3. Effects of fertilizer application on the mycorrhizal colonization of rice plants under upland conditions (assessed on the 42nd days after germination). Means followed by the common letter(s) are not significantly different at 5% level according to DMRT.

However, further dosages totally suppressed the arbuscules. In the case of superphosphate, the dosage (0.50 g/kg) decreased the arbuscular colonization whereas the higher dosage (1.00 g/kg) completely prevented their development. Effects of systemic fungicides on the growth, biomass and mycorrhizal colonization Effect of spray mode of application for two systemic fungicides on the growth of rice seedlings is presented in Table 2. The application of fungicide sprays had increased the growth of rice plants when compared to the control. Further increase in the growth was not observed in the increased dosage levels of the second and third treatments. Both the fungicides, Bavistin and Roko were found to decline the growth of rice seedlings at higher dosages. The seedlings sprayed with these fungicides became thin

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Fig. 4. Effects of systemic fungicide application on the mycorrhizal colonization of rice plants under upland conditions (assessed on the 42nd days after germination). A concentration of 2 g-fungicide/1 L-water was profusely sprayed on the seedlings. Single spray treatment received on the 10th day after germination; Double spray treatments on the 10th and 20th days; Triple spray treatments on the 10th, 20th and 30th days; The control was not treated (sprayed). Means followed by the common letter(s) are not significantly different at 5% level according to DMRT.

and weak. Fig. 4 shows the effect of these two systemic fungicides on the mycorrhizal root colonization in rice plants. The fungicide sprayed rice plants exhibited marked decrease in the percentages of total root and hyphal colonization. However, the vesicular colonization exhibited fluctuating results. In the case of arbuscular colonization, the single spray treatments showed marked decrease, and the double and triple spray treatments resulted in total suppression of the arbuscular development.

DISCUSSION Rice is a leading food crop throughout the world and its cultivation comprises 28.6% of world crop area

Table. 2. Effects of systemic fungicides application on the growth and biomass of rice plants under upland conditions (assessed on the 42nd day after germination). Number of Shoot height Root length Shoot dry weight Root dry weight Ratio of root to shoot Treatment spraying times a (cm) (cm) (mg/plant) (mg/plant) dry weight Bavistin 1 30.62 d ± 1.2 9.18 a ± 0.5 46.22 c ± 0.7 25.15 e ± 0.3 0.54 a ± 0.09 2 17.10 bc ± 0.4 8.98 a ± 0.2 32.50 b ± 0.5 24.95 e ± 0.3 0.77 c ± 0.06 3 13.54 b ± 0.1 8.20 a ± 0.3 24.99 a ± 0.6 15.05 b ± 0.3 0.60 b ± 0.05 7.38 a ± 0.5 23.17 a ± 0.4 12.71 a ± 0.3 0.55 a ± 0.08 Control 4.94 a ± 0.4 c a b f 8.72 ± 0.1 35.78 ± 0.7 26.66 ± 0.5 0.75 c ± 0.10 Roko 1 18.60 ± 1.2 2 16.70 bc ± 0.2 8.70 a ± 0.2 25.90 a ± 0.3 22.82 d ± 0.3 0.88 d ± 0.08 7.18 a ± 0.2 25.75 a ± 0.3 17.20 c ± 0.2 0.67 b ± 0.08 3 15.32 bc ± 0.6 Means followed by the common letter(s) are not significantly different at 5% level according to DMRT. Mean± SE at 1% level of significance. a A concentration of 2 g fungicide in 1 L water was profusely sprayed on the seedlings. Single spray treatment received on the 10th day after germination; Double spray treatments on the 10th and 20th days; Triple spray treatments on the 10th, 20th and 30th days; The control was not treated (sprayed).

Velu RAJESHKANNAN, et al. AMF Colonization in Upland Rice as Influenced by Agrochemical Application

under irrigation systems [10]. Aerobic upland rice is now being considered as a good water-saving model in agricultural practices [11]. Manjunath et al [12] noted for the first time that AMF colonization could occur in rice under semi-aquatic conditions and the infections increased under non-flooded conditions. However, there have been only few studies on the mycorrhizal nature in upland rice and about 20 mycorrhizal species belonging to four genera were identified [1, 13]. The rice plants under lowland cultivation are also susceptible to AMF colonization [2, 7, 14] and only semi-aquatic/ terrestrial habitat seems to be congenial for the symbiotic association. The results presented in the Fig. 1 are largely supported by literatures cited above. The critical developmental step in the life cycle of AMF colonization after the germination of rice seeds starts with the establishment of hyphal structures and later with vesicles, which ensures contact with the host root and establishment of symbiosis [15]. This showed the infectivity of AMF and the sensitivity of the plant showing little cytological reaction to AMF colonization. There might be some signaling molecules exuded by seedlings are responsible for initiation of AMF colonization [16]. The present study revealed a moderate level of root colonization in the upland rice seedlings (Fig. 2). This was greatly supported by Jalaluddin and Anwar [17] who reported that a gradual decrease in the number of spores at the seedling stage that then increased at the booting stage to reach a maximum. The occurrence of arbuscular and vesicular structures on the roots of O. sativa seedlings of all ages indicates the exchange of nutrients between AMF and the host plants. Inoculation of mycorrhizal fungi is influenced by agricultural practices [18-21], which could improve both growth and nutrient acquisition [22-25] of rice grown under upland conditions. These results are in accordance with those presented in Table 1. The observation of Phongpan and Mosier [26] contrasts that urea is not effectively utilized by rice plants because of nitrogen loss substantially and urea remained in the soil after harvest to the dry season of rice crop. It is evident that higher dosages of N and P fertilizers reduced the mycorrhizal colonization levels (Fig. 3). Besides, at the recommended dosages of N and P, the endophyte is benefited as indicated by higher

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intensities of vesicles. Dhillon and Amornpan [6] reported several beneficial effects from the application of selected inorganic fertilizers to AMF plants. A beneficial effect of AMF inoculation on biomass is frequently attributed to increased P uptake. Thomson et al [27] showed that different arbuscular mycorrhizal fungal species are inhibited to different extents by the addition of P, which typically reduces the extent of AMF formation [4-6]. However, these studies also failed to distinguish whether P inhibits the activity of AMF in the soil and the host-fungus interaction. Under aquatic environments, the requirement for minerals such as P may be met by direct absorption by either roots or shoots or both from the dissolved nutrient pool. Li et al [28] has studied the contribution of arbuscular mycorrhizal fungus Glomus caledonium on biomass [29] and yield [30] and this aspect of research deserves increased attention. Systemic fungicide spray not only reduced the extent of AMF root colonization in rice plants (Fig. 4), but also decreased the rice growth at higher dosages than the recommended level. The arbuscular structures were recorded only in the single spray treatment for both fungicides. Increasing the fungicide concentration above the recommended dosage had not increased the rice growth. This may be due to the distortion in the ecological balance created by fungicides in the rhizosphere, which has shown negative impact on plant growth. At the first site the reduction in fecundity was linked to a significant reduction in AMF infection on the sprayed plants. The fungicides applied could suppress AMF, leading to reduce resource acquisition by plants and decrease plant growth performance. The application of fungicides reduced the plant growth by acting on hyphal P transport and metabolism of AMF [30]. Dhillon [29] reported that rice plants at the nursery stage responded positively to several arbuscular mycorrhizal fungal species, and the plants showed a greater response to indigenous than non-indigenous AMF. These studies suggested that assisting colonization by inoculation of following dry nursery method might enhance the benefit of lowland rice. These fungicides could also cause induced marked alterations in the soil community structure thus disturbing the plant rhizosphere ecology [31]. The growth reduction of rice plants as a result of fungicide spray above recommended dosage may be the functional expression of the suppression of AMF

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root colonization in rice plants. The efficient use of AMF may help to achieve higher yields with optimum fertilizer dosage, and also reduce the costs and environmental pollution.

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