Abstract: We assessed how Neotyphodium coenophialum (Morgan-Jones and Gams) Glenn, Bacon and Hanlin infection influenced root characteristics of tall fescue (Lolium arundinaceum [Schreb.] SJ Darbyshire, formerly Fectuca arundinacea Schreb.) ecotypes (Fukaura, Koiwai and Showa) grown under a high (Black Andisol: high in all nutrients but low in phosphorus) and a low (Red Andisol: low in all nutrients but high in phosphorus) nutrient status soil for 30 weeks. We measured root growth parameters as well as root nutrient acquisition of infected (E+) and uninfected (E-) plants. In Black Andisol E+ plants produced more root dry matter and had greater root volume as well as root dry mass density. In Red Andisol E- plants produced more dry matter and had greater root volume as well as root dry mass density. Infected plants tended to have lower root/shoot ratio regardless of ecotypes and soils. Phosphorus and calcium (Ca) concentrations in roots of E+ plants showed higher values irrespective of soil fertility status although the values were not always statistically significant. On the other hand, concentrations of potassium (K) and magnesium (Mg) showed higher values in E- plants. In case of P, K, Ca and Mg uptake, E+ plants showed higher values than those of E- plants regardless of ecotypes grown in Black Andisol. This trend was inconsistent in E+ plants grown in Red Andisol. In this study Neotyphodium infection seems to be beneficial to tall fescue ecotypes under soil condition that is comparatively low in P but high in other nutrients content (Black Andisol) and detrimental under soil, relatively high in P but low in other nutrients content (Red Andisol).
INTRODUCTION
Tall fescue (Lolium arundinaceum [Schreb.] SJ Darbyshire), is very popular long-lived, branch forming, upright-growing cool-season forage grass with a deep root system. The massive root structure of tall fescue is frequently attributed to its wide adaptation and growth on the many different soil types (Miller, 1984; Sleper and Buckner, 1995). Tall fescue is an important cool-season perennial forage for many cattle producers in humid regions throughout the world. In recent, tall fescue has become valuable as grass cover on play areas, athletic fields, airfields, parks, highway rights-of-way and industrial sites and public institutions. Earlier reports suggest that tall fescue pastures are infected with a fungus, Neotyphodium coenophialum (Shelby and Dalrymple, 1987; Saiga et al., 2003a). Fungal endophyte-Neotyphodium coenophialum live for a significant part of their life cycle internally and asymptomatically in plants. They are unbiquitous in vascular plants, usually occurring in aboveground organs, but occasionally in roots, where they differ from mycorrhizae in that they lack external hyphae or mentels (Wilson, 1995; Saikkonen et al., 1998). It is well known that the Neotyphodium-tall fescue association is one of the mutualism. Tall fescue provides Neotyphodium with energy, nutrients, shelter and a means of propagation through the seed, while Neotyphodium provides mechanisms for improving tall fescue persistence to over grazing and insect pressure. The Neotyphodium coenophialum also confers greater drought tolerance that could improve tall fescue productivity (Bouton et al., 1993; West et al., 1993). The drought tolerance mechanisms in endophyte infected tall fescue are due to a lower net photosynthetic rate, higher stomatal resistance (Belesky et al., 1987) and greater root proliferation under drought-stressed conditions (Richards et al., 1990).
In Japan Andisols cover one-sixth of the total land surface constituting 27% of the total arable soils and 51% of the soils used for upland crops and fruit frees. Andisols of Japan characterized by low pH, P content, bulk density and high porosity with high P retention capacity and organic matter content (Agricultural Production Bureau, 1980). Phosphorus deficiency, like nitrogen deficiency, leads to an increase in root/shoot dry weight ratio. Increasing the duration of P starvation increases root dry weight and root length in particular. An increase in root surface area in deficient plants can be considered as a strategy for enhancing P acquisition from soils (Marschner, 1999). Far less is known about the influence of Neotyphodium infections on wild populations of native grass especially grown in Andisols. Despite this, the mechanisms of mineral element stress tolerance in tall fescue infected by Neotyphodium sp. is not clearly understood. Therefore, we hypothesized that E+ plants of tall fescue should produce more root biomass as well as greater nutrient acquisition in roots than E- plants grown under nutrient limited conditions and this effect would be pronounced and/or reversed when nutrient status of growing medium was more limiting. In this study we assessed how Neotyphodium infection influenced root characteristics of tall fescue ecotypes under two contrasting nutrient status soils. We measured root dry matter, root volume, root dry mass density and root nutrient acquisitions (P, K, Ca and Mg).
MATERIALS AND METHODS
Plant Materials
Three tall fescue (Lolium arundinaceum [Schreb.] SJ Darbyshire) ecotypes
viz., Fukaura, Koiwai and Showa were chosen for this experiment. Tall fescue
ecotypes of Fukaura, Koiwai and Showa infected with their naturally occurring
strains of the endophytes species N. coenophialum were collected from
Aomori, Iwate and Akita prefectures, respectively, in the northern part of Honshu
Island. To maximize the likelihood of collecting different ecotypes, plants
were collected 80-100 km apart. Endophyte-infected plants were divided into
two groups and one group was then treated with benomyl fungicide (Latch and
Christensen, 1982) to eliminate the endophyte fungus and other group was not
treated. Fungicide-treated and non-treated plants were planted in a field at
Iwate University. After a few months, the endophyte infection status was verified
by staining plant tissue with rose bengal (Saiga et al., 2003b). Plants
of the tall fescue clone for each ecotype infected with N. coenophialum
(E+) and free of endophyte (E-) were selected for the experiment. Ramets of
equal size and shape were prepared by cutting the shoots 5 cm above the crown
and the roots 5 cm below the crown. To standardize the initial size and shape
each ramet was selected from a single tiller with three adventitious roots.
Growth Condition
A pot experiment was conducted under controlled conditions since this is
the first study under Andisol with tall fescue infected with endophyte and free
of endophyte. Tall fescue plants with E+ and E- grown in two loamy Pumice and
Haplic Andisols (Black Andisol and Red Andisol, respectively) of different fertility
status. The Black Andisol was collected from the Uwadai field of Iwate University
in the prefecture Iwate while the Red Andisol was collected from the Nikaho
highland in the prefecture of Akita, northern Japan. The Black Andisol was characterized
with pH 6.02 and naturally low P content (6.71 mg 100 g-1 soil) and
high in other nutrients such as N (0.34%), K (39.6 mg 100 g-1 soil),
Ca (316 mg 100 g-1 soil), Mg (31.8 mg 100 g-1 soil), Cu
(1.42 μg 100 g-1 soil), Mn (29.4 μg 100 g-1
soil) and Zn (2.18 μg 100 g-1 soil). While the Red Andisol characterized
with pH 5.00 and naturally high content of P (15.3 mg 100 g-1 soil)
and low in other nutrients such as N (0.12%), K (20.3 mg 100 g-1
soil), Ca (0.91 mg 100 g-1 soil), Mg (1.18 mg 100 g-1
soil), Cu (0.36 μg 100 g-1 soil), Mn (3.20 μg 100 g-1
soil) and Zn (0.10 μg 100 g-1 soil). Despite Black Andisol was
higher in P-retention capacity (1962) with higher organic matter content (184
g kg-1) compared to Red Andisol having lower in P-retention (604)
and organic matter content (94 g kg-1). After removing plant debris
the air-dried soils were passed through a 4 mm sieve and the pots were filled
with this soil (2 kg pot-1). Water was then added to bring the soil
to Field Capacity (FC). The tops of the pots were covered with a black polyethylene
film to prevent evaporation and the soils were allowed to equilibrate at glasshouse
temperature. The pots were weighed prior to transplanting the plants into them.
The plants were grown in a control condition (Rahman and Saiga, 2005; Rahman
et al., 2006) for 30 weeks without application of any chemical fertilizers.
Pots were maintained at field capacity (pF 2) throughout the experiment
by adding tap water as required.
Harvest and Mineral Analysis
The plants were allowed to grow for 30 weeks and then plants were separated
from soil, by washing the soil over a screen surface. The roots were separated
and stored at low temperature until the measurement of root volume. Roots and
shoots were dried at 80°C for 48 h in a forced-air oven and weighed to obtained
dry yield. Root/shoot ratio and root dry mass weight were calculated. Dried
root samples were digested with HNO3-HClO4 acid (2:1)
and concentrations of K, Ca and Mg were measured by atomic absorption spectrophotometry
(Perkin Elmer-3300). The concentration of P was measured colorimetrically using
a vanadomolybdate-phosphoric yellow color method (Jasco Ubest-30 UV/VIS spectrophotometer)
as described by Jackson (1973). From the concentration of different nutrients
in roots, nutrients uptake per plant were calculated.
Experimental Design
The experiment was set up as a split plot design with two soil fertility
levels (main plots) and endophyte/tall fescue ecotype associations (split plots)
as random effects replicated three times.
Statistical Analysis
A three-way analysis of variance (ANOVA) was employed to determine whether
endophyte infection affected root growth and nutrient concentration as well
as uptake. The ANOVA model included endophyte, soil and ecotype as the mail
source of variation and three pairwise: endophyte x soil, endophyte x ecotype
and soil x ecotype and one three-way: endophyte x soil x ecotype interaction.
Multiple range tests were conducted using the method of RYAN-EINOT-GABRIEL-WELSCH
(Ryan, 1960) to compare results for endophyte infection with the variables at
a 5% level of significance. The Statistical Analysis System, Version 6 (SAS
Inst., Cary, NC, USA) was used for all analysis. Means between plants grown
in various soils were also compared by Least Significant Difference (LSD) test
(p<0.05).
RESULTS AND DISCUSSION
Analysis of variance revealed that the root dry weight was significantly affected by endophyte infection, soil fertility, plant ecotype, endophyte x soil, endophyte x ecotype and soil x ecotype but the interaction of endophyte x soil x ecotype was not statistically significant (Table 1). Significant effect of endophyte infection, soil fertility, plant ecotype, endophyte x soil, endophyte x ecotype and soil x ecotype on root volume as well as root dry mass density was observed but the interaction of endophyte x soil x ecotype was not statistically significant.
| Table 1: | ANOVA test for the root characteristics of tall fescue |
| Table 2: | Root characteristics of tall fescue ecotypes as affected by endophyte infection in different Andisols |
| *Values within the rows and parameters for each variable with the same letter(s) are not significantly different at p<0.05; **LSD (p<0.05) between Black Andisol and Red Andisol (columns) | |
Root/shoot ratio was significantly affected by all the sources of variations except interaction of endophyte x ecotype and endophyte x soil x ecotype.
In Black Andisol, root dry weight was higher in E+ plants than in E- plants (Table 2). The opposite was observed in Red Andisol. Although in Black Andisol, ecotype Fukaura and in Red Andisol, ecotype Showa did not show any significant differences on root dry weight between E+ and E- plants. Endophyte infection reflected positive effects on root volume as well as root dry mass density in Black Andisol while it was negative in Red Andisol for all forage tall fescue ecotypes although the values were not always statistically significant. Malinowski and Belesky (1999) observed that endophyte infection increased 57% root dry matter as compared to non-infected tall fescue grown in Al-free nutrient solution while Al in growth medium depressed dry matter accumulation in roots by 40% of E+ plants and 3% of E- plants. Lui et al. (1996) found that E+ fine fescues (Festuca sp.) had relatively less reduction in root DM than did E- plants when grown in an acidic soil or sand and that endophyte infection had no effect when plants were grown in nutrient solutions. Malinowski et al. (1998) also found that with increased P availability in soil, root dry matter in E+ plants of tall fescue were significantly less when compared to E- plants. They inferred that endophyte-infected tall fescue is not responsive to high soil P indicating that an interaction between endophyte infection and abiotic factors exists.
All tall fescue ecotypes showed significantly lower root/shoot ratios for E+ plants than that of E- plants under Black Andisol. While under Red Andisol ecotype Fukaura and Koiwai showed lower values and Showa showed higher values in E+ plants indicating ecotype behaved differently with soil fertility. Regardless of ecotype, E+ plants showed 18.33% higher root/shoot ratios as compared to E- plants. This increase is attributable to the higher rates of development of roots as compared to shoots in E+ plants than that of E- plants. Malinowski and Belesky (1999) observed that endophyte infection did not affect root/shoot ratio of plants grown in Al- nutrient solution. In contrast, E+ plants tended towards a reduced root/shoot ratio when compared to E- plants in Al+ growing conditions.
With a few exceptions mineral concentration and accumulation in roots was strongly influenced by endophyte status, soil and tall fescue ecotype (Table 3). Phosphorus, K and Ca concentrations in roots of forage tall fescue were affected endophyte x soil x ecotype interactions.
| Table 3: | ANOVA test for the root nutrient elements in tall fescue |
| Table 4: | Metal macro-nutrient acquisition in root of tall fescue as affected by endophyte infection in different Andisols |
| *Values within the rows and parameters for each variable with the same letter(s) are not significantly different at p<0.05; **LSD (p<0.05) between Black Andisol and Red Andisol (columns) | |
Higher P and K uptake was observed in E+ plants grown in Black Andisol whereas lower was observed in E+ plants grown in Red Andisol (Table 4). Endophyte-infected plants had significantly higher root P concentrations than those of E- plants irrespective of soils whereas K concentration in root was substantially higher in E- plants than E+ plants grown in both the soils. Differences in Ca and Mg concentration between E+ plants and E- plants grown in both the soils were not statistically significant although higher amount of Ca was observed in E+ plants. Higher amount in root Ca concentration between E+ and E- plants could suggest an influence of the endophyte infection status upon the cell wall formation since Ca makes an electrical bridge between carboxyl group and pectin chain (Rahman, 1997) which may be responsible for wide adaptability of E+ plants in biotic and abiotic stress.
CONCLUSIONS
Our results indicated that, as compared to E- plants, E+ plants of tall fescue had better performance at high nutrients element level, but significantly lower performance at low nutrients element level, which presumably reflects the balance between the advantage of infection and the cost to the host supporting the endophyte. The variation in root characteristics of forage tall fescue ecotypes may be caused by chemical signaling system in E+ plants as Neotyphodium sp. are not exists in plant roots.