African Journal of Microbiology Research Vol. 6(11), pp. 2779-2784, 23 March, 2012 Available online at http://www.academicjournals.org/AJMR DOI: 10.5897/AJMR11.1456 ISSN 1996-0808 ©2012 Academic Journals

Full Length Research Paper

Development status of Arbuscular mycorrhizal fungi associated with invasive grandiflora Hogg.

Bai Yanfang1,2, Li Min1,2 and Guo Shaoxia1,2*

1Institute of Mycorrhizal Biotechnology, Qingdao Agricultural University, Qingdao Shandong Province 266109, China. 2College of Garden and Horticulture, Qingdao Agricultural University, Qingdao Shandong Province 266109, China.

Accepted 23 January, 2012

According to the distribution of Coreopsis grandiflora Hogg., samples were collected from two natural invasion sites; Jufeng and Beijiushui and cultivated site to research AM fungal arbuscule structure of C. grandiflora Hogg., colonization status in different sample plots and altitudes, and the correlation between colonization status and soil factors in the rhizosphere of C. grandiflora. The result showed that C. grandiflora could form heavy Paris-type arbuscule structure, Beijiushui mycorrhizal colonization percentage and numbers of entry points was highest and significant differences with Jufeng and campus. C. grandiflora Hogg. mycorrhizal colonization percentage was on the rise with rising altitudes. In addition to altitudes, soil factors as well as to influence AM fungal colonization status, there were very positive correlation (r=0.792) and positive correlation (r=0.725) between mycorrhizal colonization percentage and the content of organic carbon, available potassium in soil, respectively; there were very positive correlation (r=0.854) and very negative correlation (r=-0.966) between numbers of vesicles P- type arbuscule) and the content of available potassium, pH in soil; there were negative correlation (r=- 0.782) between numbers of entry points and the content of available phosphorus. High infection rate and the formation of typical arbuscular form of C. grandiflora Hogg. at Laoshan should have an important role on the process from the invasion to becoming constructive .

Key words: Coreopsis grandiflora, Arbuscular mycorrhiza fungi (AM fungi), mycorhizae structure, soil factors.

INTRODUCTION

Arbuscular mycorrhiza fungi (AM) are major component Compositae are mostly focused on the common of soil microbial communities in terrestrial ecosystems, cultivated crops, such as Helianthus annus and wild which can form mutually beneficial symbiosis with 90% of Neopallasia pectinata, Artemisia argyi, A. eriopoda vascular plants (Liu and Chen, 2007). A mycorrhizal and Taraxacum mongolicum (Muthukumar and structure, such as infection conditions, mycelial growth Tamilselvi, 2010; Bao and Yan, 2004) study on and development, is the material basis of function of arbuscular type of invasive plants in has been arbuscular mycorrhizal symbiosis. Gallaud firstly rarely reported. In addition, the ability of AM fungi to form proposed two types of arbuscular mycorrhizal in 1905, symbiotic associations with host plants and mycorrhizal later arbuscular mycorrhizal were divided into three effects are relative to many environmental factors, types: Arum-type (A-type), Paris-type (P- type) and including elevation and soil factors. The significant Intermediate-types (I- type) (Smith and Smith, 1997). changes of altitudes, temperatures and precipitation have Today arbuscular mycorrhizal studies of important implications on plant growth and AM fungi infection (Cai et al., 2005). Supply of soil nutrients plays an important role on mycorrhizal infection and the formation of the structure of AM fungi; currently the *Corresponding author. E-mail: [email protected]. effects of the elevation and soil nutrients on the structure 2780 Afr. J. Microbiol. Res.

formation and growth of AM fungi have not reached the staining solution at room temperature overnight; observed the same result, and still lack a comprehensive and samples with an optical microscope after separation by lactic acid. systematic study (Feng, 2003; He et al., 2010). We determined the morphology of mycorrhizae (arbuscular, vesicles and hyphae) according to Smith and Smith (1997) method; C. grandiflora Hogg., which has been identified as calculated the numbers of laps, vesicles and penetrated several invasive species in China (Xu and Qiang, 2004) has points of mycelium of 100 root segments from every sampling sites. spreaded in Jufeng of Laoshan and Beijiushui and Mycorrhizal infection rate was calculated with the root segment become a serious threat to local ecosystems. Walling and frequency conventional method of Biermann and Linderman (1981), Zabinski (2006) proved that the hyphae in rhizosphere of as the following formula: invasive plant Centaurea maculosa was higher than native plants compared with the indigenous plant, roots of Sapium sebiferum are rich in AMF (Nijjer et al., 2008); high infection rate of arbuscular has an important role on the process from the invasion to becoming constructive Photographed using Olympus BX51 microscope during the process species. In this paper, AM fungal colonization in C. of observation. grandiflora Hogg. from Laoshan region was studied preliminarily, which will provide a theoretical basis for Determination of soil factors research and control of this invasion plant. Content of alkali-hydrolyzed nitrogen in soil was determined with Alkali-diffusion method, soil available P was determined by sodium

bicarbonate - Mo-Sb colorimetry, soil available K with ammonium MATERIALS AND METHODS acetate - flame photometric method, soil organic matter with

Natural conditions of experimental site potassium dichromate oxidation method and soil pH with potential method (Lu, 2000). Laoshan of Qingdao city brink of the Yellow Sea belongs to a temperate maritime climate,where the average annual temperature is 12.6°C, the average annual rainfall is 700 ~ 800 mm, and frost- Data processing free period is 180 ~ 200 days. The soil of experimental site is brown soil and slightly acidic. Vegetation is mainly Pinus thunbergii, Experimental data were analyzed with SPASS17.0 software for Pinus densiflora, Robinia pseudoacacia, Ailanthus altissima, one-way ANOVA and correlation analysis. Pistacia chinensis and Ulmus macrocarpa; Spiraea shrub species mainly includes Spiraea fritschiana, Prunus japonica, Zanthoxylum bungeanum, Spiraea trilobata, Rubus corchorifolius RESULTS AND ANALYSIS and Lauraceae obtusiloba; Herbaceous species mainly includes Arrhenatherum elatius, Adenophora axilliflora, Lilium Mycorrhizal structures tsingtauense, Veratrum nigrum, Thalictrum minus var. hypoleucum and Polygonatum odoratum. The results showed that C. grandiflora Hogg. can form good arbuscular mycorrhizae, whose surface structure Sample collection included intrusion point (Plate 1 of Figure 1), hyphae According to the distribution of C. grandiflora Hogg., samples were outside the root (Plates 2 to 4 of Figure 1) and spores collected from two natural invasion sites Jufeng (Sunrise side of outside the root (Plate 9 of Figure 1) etc., and the internal Laoshan Mountain, Altitude 12 ~ 426 m) and Beijiushui (nightside of structure included hyphae (Plate 5 of Figure 1), Laoshan Mountain, Altitude 81～450 m) and cultivated site arbuscular (Plates 6 to 8 of Figure 1) and vesicles (Plates (Qingdao Agricultural University campus). Removed the topsoil, 10 to 12 of Figure 1). Arbuscular types in roots of C. take the roots and rhizosphere soil of C. grandiflora Hogg., samples grandiflora Hogg. are mainly P-type, whose arbuscular were placed in ziplock bag and registered, recorded collection time and altitude of sampling location by GPS. Each sample point was structure is mainly mycelium ring (Plates 6 to 8 of Figure set 3 to 4 replicates. 1). Vesicles had several shapes with pear shape, round Samples were taken back to the laboratory, the roots were put shape, oval shape, long oval shape, spindle shape and into the refrigerator for determination of mycorrhizal colonization; irregular shape, most of which are elliptical shape and and soil samples were air-dried and screened after a 20 mesh sieve oval shape, whose size is 14.76 ~ 98.4 μm. in the laboratory for determination of soil chemical properties.

Observation of mycorrhizal morphology Development status of AM fungi in rhizosphere of C. Mycorrhizal morphology was observed by acid fuchsin staining grandiflora Hogg. (Kormanik et al.,1980), plant roots were mixed every sampling point at one altitude, rinsed with distilled water and cut into 0.5 ~ 1.0 cm Development status of AM fungi from different sites segments, which were put into test tubes with 5 ~ 10% KOH solution, then the tubes were put into water bath pot at 90°C for 15 Colonization of AM fungi from different sites was showed min, then rinsed with distilled water and soaked in 2% HCL solution for 5 min; removed the solution and added 0.01% acid fuchsin lactic in Figure 2. Mycorrhizal infection rate of C. grandiflora glycerol. Hogg. From Beijiushui is highest, an average of 51.4%, Yanfang et al. 2781

Figure 1. Mycorrhizal structure of Coreopsis grandiflora; 1. Entry points (EP); 2, 3, 4. External hyphae (EH); 5. Internal hypha (IH); 6, 7, 8. Paris arbuscule (A); 9. Spore (S); 10, 11, 12. Vesicle (V) Bars: 1, 2, 3, 4, 5, 9, 10 mean 100 µm; 6, 7, 8, 11, 12 mean 50 µm.

60

a Beijiushui 50 Jufeng Campus 40 b b a 30

20 ab

a a 10 a a b b b 0 MCP（%） NHC（No./100mm） NV (No./100mm) NEP (No./100mm)

Figure 2. Development status of Arbuscular mycorrhizal fungi of Coreopsis grandiflora in different sample plots. LSD multiple comparisons in difference sample plots, significant differences in each column are indicated by different letters (P≤0.05). MCP=Mycorrhizal colonization percentage, NHC= Numbers of hyphal coils, NV=Numbers of vesicles, NEP=Numbers of entry points. 2782 Afr. J. Microbiol. Res.

120

Jufeng Beijiushui

） 100

%

（

80

60

40

Mycorrhiza collonization percenta(%) 20 mycorrhizal colonization percenta percenta colonization mycorrhizal

0 0 50 100 150 200 250 300 350 400 450 500

altitudesAltitudes （ (m)m)

Figure 3. Arbuscular mycorrhiza fungi colonization status in roots of Coreopsis grandiflora in different altitudes.

Table 1. Correlation analysis between AM fungi development minimum is 8.3% at an altitude of 12 m; maximum of status and soil factors in rhizosphere of Coreopsis grandiflora. infection rate in Beijiushui site is 74.7% at altitude from 337 to 348 m; minimum is 15.5% at an altitude of 144 m Altitudes NEP NHC NV (Figure 3). MCP 0.485 -0.036 -0.545 -0.349 Correlation analysis showed that the infection rate and NV -0.405 -0.181 0.842** altitude was positive correlated and numbers of vesicles, NEC -0.328 -0.147 invasive point and hyphae turns was negative correlated NEP -0.232 to altitude, but both neither reached a significant level. In

** addition, only numbers of hyphae laps and vesicles were means correlation coefficients significant at P<0.01 (2-tailed). significantly positively correlated (r=0.842), the other

indicators had no significant correlation (Table 1).

which was significantly higher than Jufeng and campus; The effects of soil factors on development status of mycorrhizal infection rate from Jufeng (30.2%) was AM fungal higher than from campus, but the difference was not significant. The numbers of vesicles in roots from three The results showed that infection rate was significantly sites were significantly different, Jufeng 29.60/100 mm, positively correlated to soil available potassium (r=0.725), Beijiushui 16.08/100 mm and the campus only 4.90/100 and highly significantly positively correlated to soil mm. The numbers of intrusion points of 3 sites were also organic matter content (r=0.792); the number of mycelium significantly different, Beijiushui 9.31/100 mm, Jufeng ring was significantly positively correlated to the content 4.35/100 mm and campus 2.29/100 mm, but mycelium of available potassium (r=0.854), and was highly rings had no significant difference between each other, significantly negatively correlated to soil pH (r=-0.966); Beijiushui 7.58/100 mm, Jufeng 7.51/100 mm and the number of intrusion points was significantly negatively campus 3.18/100 mm. correlated to soil available phosphorus content (r=-0.782) (Table 2).

The impact of altitude on development status of AM fungal and correlation analysis DISCUSSION AND CONCLUSION

Mycorrhizal infection rate of C. grandiflora Hogg. The results of this study showed that C. grandiflora Hogg. increased with the altitude upward. Maximum of infection has a strong symbiotic relationship with mycorrhizal fungi, rate in Jufeng site is 95.6% at altitude from 339 to 426 m; a large number of mycorrhizae can replace roots for Yanfang et al. 2783

Table 2. Correlation analysis between AM fungi development status and soil factors in rhizosphere of Coreopsis grandiflora.

Available N Available P Available K Organic carbon pH MCP 0.277 -0.489 0.725* 0.792** -0.577 NV 0.574 -0.226 0.344 0.23 -0.367 NHC 0.056 -0.417 0.854** 0.43 -0.966** NEP -0.150 -0.782* 0.319 0.193 -0.631

*means correlation coefficients significant at P<0.05; ** means correlation coefficients significant at P<0.01 (2-tailed).

absorption and competition for nutrients from the near highly significantly negatively correlated, proved that high native plants to result imbalance of plant transfer of phosphorus content can inhibit formation of intrusion nutrients (Berta et al., 1993; Vance et al., 2003; Carey et points and growth of hyphae outside the root. Mycorrhizal al., 2004). However, Pringle et al. (2009) proved that non- growth and development requires a certain amount of mycorrhizal invasive plants are also able to successfully potassium, an appropriate increase in potassium content invade new habitat (Ahulu et al., 2005). For example, is beneficial for the infection and the symbiotic formation Callaway et al. (2008) have found that the secretions of of AM fungi (Li et al., 2002). Soil available K content was non-mycorrhizal onion mustard after invasion in North significantly positively related to mycorrhizal infection rate America disrupted the symbiotic relationship of native of C. grandiflora Hogg. and had the greatest impact on plants and AM fungi plant invasion, which reduced the arbuscular. Correlation study is a preliminary exploration growth and competitiveness of native plants, and of the impact of a single soil factors on mycorrhizal ultimately produced a positive feedback on invasive colonization status, there may be some interaction to plants. This study found that arbuscular of C. grandiflora mycorrhizae form between the soil factors, which needs Hogg. is P-type, that is, the AM fungi forms the typical to be furtherly studied. mycelium root ring structure within the root. Cavagnaro et In conclusion, C. grandiflora Hogg. can form a good al. (2001) believed that the formation of P-type arbuscular symbiotic relationship with local AM fungi after invasion. takes longer time than the A-type, because mycelium root The arbuscular mycorrhizal (AM) fungi are common ring formation take time. Morphology of A-type and P- components in terrestrial ecosystems, it is important for type is relative to groups of plant succession, from the the control of invasive plants to study AM fungi Pioneer group, early succession group to late succession morphology of invasive plants and its correlation with group, the ratio of A-type arbuscular mycorrhizal reduced environmental factors. in the order and the P-type arbuscular mycorrhizal increased in the order (Ahulu et al., 2005). Imhof and Weber (1997) proved that P-type structure is a relatively ACKNOWLEDGEMENT advanced type at stress and low nutrient conditions because mycelium root ring may be a signal controlled by This research was supported by Natural Science plant. Brundrett and Kendrick (1990a and b) speculated Foundation of China (No. 30471164 and 40171049) and that the slow P-type AM colonization was beneficial for also supported by Natural Science Foundation of mycorrhizal host plants to maintain a low energy supply, Qingdao City (08-1-3-20-jch). which is conducive to growth and development of plant. C. grandiflora Hogg. is a perennial herb and has longer growth cycle, so the longer invasion period and mycelial REFERENCES growth period provide the basis for the formation of mycelium root ring. The formation of P-type arbuscular is Ahulu EM, Nakata M, Nonaka M (2005). Arum- and Paris-type arbuscular mycorrhizas in a mixed pine forest on sand dune soil in favorable for the growth and competition of C. grandiflora Niigata Prefecture, central Honshu, Japan. Mycorrhiza, 15: 129-136. Hogg. in the invasion habitat. Bao YY, Yan W (2004). Arbuscular mycorrhizae and their structural This study found that soil organic matter content and types on common plants in Grasslands of mid-western Inner the infection rate has a significant positive correlation, Mongolia. Biodivers. Sci., 12(5): 501-508. Berta G, Fusconi A, Trotta A (1993). VA mycorrhizal infection and the indicating that soil organic matter content is conducive to morphology and function of root systems. Environ. Exp. Bot., 33: 159- the development of mycorrhizal fungi; and soil pH was 173. negatively correlated to mycorrhizal infection rate and Biermann BJ, Linderman RJ (1981). Quantifying vesicular-arbuscular significantly influenced the formation of P-type mycorrhizae: a proposed method toward standardization. New Phytol., 87: 63-67. arbuscular, which is consistent with the results of Brundrett MC, Kendrick B (1990a). The roots and mycorrhizas of previous research (He et al., 2005). Nitrogen and herbaceous woodland plants. I. Quantitative aspects of morphology. phosphorus contents were negatively correlated with the New Phytol., 114: 457-468 invasion points, especially phosphorus content was Brundrett MC, Kendrick B (1990b). The roots and mycorrhizas of 2784 Afr. J. Microbiol. Res.

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