Proceedings of The Fourth International Iran & Russia Conference 327

Role of Endophytic Fungi in Forage Production of Tall Fescue, arundinacea

Mohammad Reza Sabzalian1, Reza Mohammadi2 and AghaFakhr Mirlohi 3

1, 2, 3-Department of Agronomy and Breeding, College of Agriculture, Isfahan University of Technology, Phone: 0311-391-3450, Fax: 0311-391-2254, E-mail: [email protected]

Abstract Symbiotic relationship has been found between endophytic fungi and most cool- season grasses including 80 genera and 100 species of subfamily . In this relation, endophytic fungi gain their food and energy from host and instead improve host characteristics such as yield and resistance to intense grazing and biotic and abiotic stresses. These effects induced from endophytic fungi can increase net production of plant forage. Six genotypes of tall fescue, Festuca arundinacea, were used in this research to evaluate endophytic fungi role in forage production. Endophyte-free versions of each genotype were prepared using a fungicide mixture of Fulicor and Propiconazol from endophyte-infected plants. These genotypes were planted in a randomized complete block design with three replications in the field. Fresh and dry weight of forage produced, tiller number and rate of re-growth of each genotype (endophyte-infected and endophyte-free versions) were measured after eight months. Results of this study showed that endophytic fungi could increase fresh and dry weight of plant forage. Endophyte-infected plants had two to ten times more tiller number than endophyte-free counterparts. Endophyte also enhanced re-growth of infected plants after clipping. This may be due to allocation of more assimilates to plant roots. This study showed that endophytic fungi can improve production of plant forage and may be used in other grass species important for forage production.

Key words: endophyte, Festuca arundinacea, forage plant,

Introduction Tall fescue (Festuca arundinacea Schreb.) is infected by the Neotyphodium coenophialum Glenn, Bacon, Hanlin. This fungus spends its life cycle within the plant without any external sign of infection (Siegel et al., 1985). Hyphae of Neotyphodium are distributed in all plant parts except plant roots (Bacon et al., 1977; Clark et al., 1983). This association of tall fescue and its endophyte has been suggested to be a mutualistic symbiosis in which, grass benefits by increased growth, deterrence of insects and mammalian herbivores, and tolerance to stress environments while the fungus receives nutrients from the plant apoplast, reproduce and disseminate via seed production (Bacon and Siegel, 1988). Endophyte infection has increased tillering and herbage growth in tall fescue clones (Belesky et al., 1987). At the population level, infected tall fescue seedlings, showed greater germination and tiller and dry weight production than noninfected counterparts (Clay, 1987). Results on the effect of endophyte on these traits in the field, however, are not crucial and involve some contrasting reports. Siegel et al. (1984) found no difference in survival rate and herbage yield between infected and noninfected populations under well adapted condition. By contrast, in stressful environment, Read and Camp (1986) reported enhanced growth and better survival of infected plants. The objective of this study was to determine the effects of the

327 Proceedings of The Fourth International Iran & Russia Conference 328 endophyte association on forage production and associated traits in a broader range of tall fescue genotypes.

Materials and Methods Plant Materials Six tall fescue genotypes clonally propagated were used for this study. Seeds of three accessions were originally planted in the greenhouse and six compatible host- endophyte combinations from accessions were selected. Microscopic examination of leaf sheaths confirmed infection of plants and compatible combination was chosen based on high hyphae concentration and its unbranched hyphae movement. Each selected plant was separated into two groups of individual tillers which were transplanted into separate plots in the field. One plot of each plant (genotype) was treated (sprayed) with Propiconazole [1-(2-(2-4-dichlorophenyl)-4-propyl-1, 3- dioxolan-2-y1) methyl-1H-1, 2, 4-triazole] and Folicur as a fungicide mixture at 2a.i. (active ingredient) and 1ml per liter, respectively. The fungicide treatment was repeated two times, 7d apart. Microscopic examination of new tillers produced in the treated plots confirmed eradication of the endophyte. New tillers of endophyte- infected and endophyte-free plants were transplanted to experimental field. The experimental design was a randomized complete block with a factorial arrangement of the treatments (six genotypes and their infection status) with 3 replications including 6 hills of plant per replication. Each hill comprised of approximately 5 tillers. Plots were 1.5 × 1.5 m2 in size and contained a rich clay-loam soil. Throughout the experiment, all plots were watered twice a week, and fertilized (75 kg/ha N) before flowering stage in the spring. Plant Analysis After 8 months of field growth, plants were cut from 5 cm of ground level, oven dried at 60°C for 48 h and weighted. Shoot fresh and dry weight of one hill was also measured. One hill from each plot was randomly selected, and number of tillers per hill was measured. After that, the hill was washed free of soil, roots were separated, weighted and oven dried at 60°C for 48 h and weighted again to measure fresh and dry matter of root. After two weeks of cutting, the height of regrowth in each plot was measured. Analyses of variance were performed for each variable and treatment means were compared using Duncan’s multiple range test.

Results The experimental plant genotypes used in this study showed significant (P<0.01) differences for all variables. When pooled across genotypes, the variables including shoot fresh and dry weight, root fresh and dry weight, plot herbage yield, numbers of tillers per hill and regrowth height after cutting were significantly (P<0.01) increased by endophyte infection (Figure 1) but the increase rate among genotypes was different and in some cases endophyte-free genotypes had greater value of the trait than their endophyte-free counterparts. This was resulted in significant (P<0.01) interaction between genotype and endophyte infection for shoot and root weight (fresh and dry), numbers of tillers per hill and regrowth height but not for plot herbage yield (Table 1).

Discussion Tiller number per hill, herbage growth, and root fresh and dry yield were increased in infected clones (Fig. 1). Similar results in tillering and herbage production have been obtained with infected and noninfected clones of tall fescue and perennial rye grass (De Battista et al., 1990; Arechavaleta et al., 1989; Belesky et al., 1987). Infected

328 Proceedings of The Fourth International Iran & Russia Conference 329 plants produced (P<0.01) more root dry matter than endophyte free counterparts. Enhanced root mass was also reported in endophyte-infected clones of perennial ryegrass (Latch et al., 1985). In this study, it is showed that endophyte infection my decrease growth of at least some tall fescue genotypes, but it is not clear how the endophytic fungus may alter the plant’s physiology to achieve these differences. Change in hormonal balance of the host plant could be a possible fungal mechanism to alter host growth (Arachevaleta et al., 1989; Belesky et al., 1987). It has been established that the endophyte of tall fescue produces IAA in culture (De Battista, 1989), But, the effect on IAA in the plant are still unknown. The high interactions of endophyte infection and plant genotype may result from specific relationships between each plant and fungus genotype. This effect implies genotypic differences among individuals of both plant and fungus. Greater regrowth after cutting in endophyte-infected plants, suggests that endophyte infected plants were more efficient in using supplies of root for shoot regrowth. This effect may also relate to alteration in plant physiology and hormonal balance. In summary, endophyte infection may increase root growth and enhance forge production of tall fescue host plant, but because of the presence of interactions between endophyte and grass genotypes, the same effect may not be extrapolated to other genotypes of the endophyte–harboring plant species. More information is needed on the physiological bases and mechanisms by which fungal endophyte affect host growth characteristics, along with the effect of environmental factors on the expression of them before a generalized conclusion.

References 1- Arechavaleta M, Bacon CW, Hoveland CS,, Radcliffe DE,(1989) Effect of the tall fescue endophyte on plant response to environmental stress. Agron J. 81:83-90. 2- Bacon CW, Porter JK, Robbines JD, Luttrell ES(1977) Epichlo typhina from toxic fescue grasses.Appl. Environ. Microbiol. 34:576-581. 3- Bacon CW, Siegel MR (1988) The endophyte of tall fescue. J. Prod. Agric. 1:45-55. 4- Belesky DP, Devine OJ, Pallas JE, Stringer WC(1987) Photosynthetic activity of tall fescue as influenced by a fungal endophyte. Photosynthet. 21:82-87. 5- Clark EM, White JF, Patterson RM (1983) Improved histochemical techniques for the detection of Acremonium coenophialum in tall fescue and methods in vitro culture of the fungus. J. Microbiol. Methods 1:149-155. 6- Clay K (1987) Effects of fungal endophytes on the seed and seedling biology of perenne and Festuca arundinacea. Oecologia 73:358-362. 7- De Battista JP (1989) Evaluation of tall fescue production in the greenhouse and effect of endophyte infection on rhizome expression. M.S. thesis. Univer. Of Georgia. Athens. 8- De Battista JP, Bouton JH, Bacon CW, Siegel MR (1990) Rhizome and herbage production of endophyte-removed tall fescue clones and population. Agron. J. 82:651- 654. 9- Latch GCM, Hunt WF, Musgrave DR (1985) Endophytic fungi affect growth of perennial ryegrass. N.Z.J. Agric. Res. 28:165-168. 10- Read JC, Camp BJ (1986) The effect of fungal endophyte Acremonium coenophiallum in tall fescue on animal performance, toxicity and stand maintenance. Agron J. 78:848-850. 11- Siegel MC, Latch GCM, Johnson MC (1985) Acremonium fungal endophytes of tall fescue and perennial ryegrass: significance and control. Plant Dis. 69:179-183. 12- Siegel MR, Johnson MC, Varney DR, Nesmith WC, Buckner RC, Bush LP, Burrus PB, Jones TA, Boling JA(1984) A fungal endophyte in tall fescue: incidence and dissemination. Phytopathology 74:932-937.

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Table 1-Analysis of variances for tall fescue forage production traits in endophyte-infected and noninfected plant genotypes. source df SFW SDW RFW RDW PHY TIL REG

Genotype 5 ** ** ** ** ** ** **

Endophyte 1 ** ** ** ** ** ** **

Genotype*Endophyte 5 ** ** ** ** NS ** **

**, NS Significant at the 0.01 level and Not Significant, respectively. SFW: Shoot Fresh Weight, SDW: Shoot Dry Weight, RFW: Root Fresh Weight, RDW: Root Dry Weight, PHY: Plot Herbage Yield, TIL: Tiller numbers per hill and REG: Regrowth height.

1400 a 1200

1000 b a Weight 800 E+ (gr) 600 a a b E- b b 400 a 200 b

0 SFW SDW RFW RDW PHY Trait

Figure1(a)- Shoot fresh weight (SFW), shoot dry weight (SDW), root fresh weight (RFW), root dry weight (RDW) and plot herbage yield (PHY) of tall fescue clones as affected by endophyte infection. Values are means of six clones and three replications per clone. Means with the different letter are significantly different.

300 20 a a 250 b 15 200 E+ E+ 150 b 10 E- E- 100

Height (cm) Height 5 Tiller number 50 0 0 TIL REG

330 Proceedings of The Fourth International Iran & Russia Conference 331

Figure1(b)- Tiller number per hill and mean regrowth height of tall fescue clones as affected by endophyte infection. Values are means of six clones and three replications per each clone. Means with the different letter are significantly different. TIL:Tiller number and REG: Regrowth

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