R ESEARCH A RTICLE doi: /scienceasia Somchit Youpensuk a, *, Sittichai Lordkaew b, Benjavan Rerkasem c

Transcription

1 R ESEARCH A RTICLE doi: /sciencesi ScienceAsi 34 (2008): Arbusculr mycorrhizl fungi ssocited with tngerine (Citrus reticult) in Ching Mi province, northern Thilnd, nd their effects on the host plnt Somchit Youpensuk, *, Sittichi Lordkew b, Benjvn Rerksem c Deprtment of Biology, Fculty of Science, Ching Mi University, Ching Mi 50200, Thilnd b Multiple Cropping Centre, Fculty of Agriculture, Ching Mi University, Ching Mi 50200, Thilnd c Deprtment of Agronomy, Fculty of Agriculture, Ching Mi University, Ching Mi 50200, Thilnd * Corresponding uthor, e-mil: scboi027@chingmi.c.th ABSTRACT: There re mny tngerine (Citrus reticult) orchrds in northern Thilnd. These orchrds re supplied with different levels of fertilizers. The objective of this study is to investigte rbusculr mycorrhizl (AM) fungi ssocited with tngerine in Ching Mi province, northern Thilnd, nd the effect of AM fungi on the growth of the ir lyered tngerine vriety Si Num Phung with different levels of nitrogen (N) nd phosphorus (P) fertilizers in pot experiment. Percentge of AM coloniztion in the tngerine roots nd spore density in the rhizosphere vried significntly with the vilble P levels in the orchrd soil. Mens of root coloniztion nd spore density were significntly depressed t >500 mg P/kg soil. Twenty-two species of AM fungi were found to be ssocited with tngerine in orchrds of the Ching Mi province. The effects of AM fungi, nd N nd P fertilizers on ir lyered tngerine plnts were investigted in pots for ten months. AM fungi incresed growth of the host plnt especilly in pots with N but without P fertilizers. AM fungi incresed concentrtions of P nd Mg in leves of tngerine. Appliction of N nd P fertilizers depressed root coloniztion of AM fungi in the pot experiment. This study hs shown tht wide rnge of AM fungi is ssocited with tngerine in commercil orchrds, but with high levels of N nd P fertilizers the increse in growth of tngerine trees due to the ssocition with AM fungi my be limited. KEYWORDS: rbusculr mycorrhizl fungi, tngerine, phosphorus Received 24 Sep 2007 Accepted 24 Jun 2008 INTRODUCTION Mycorrhizs re symbiotic ssocitions between some soil fungi nd plnt roots. The host plnt receives minerl nutrients while the fungus obtins photosyntheticlly derived crbon compounds. The most bundnt ssocition is rbusculr mycorrhiz 1. Arbusculr mycorrhizl (AM) ssocitions form when host roots nd comptible AM fungi re both ctive in close proximity nd the soil conditions re fvourble 1. AM fungi produce rbuscules for nutrient interchnge with the host inside corticl cells nd estblish diffuse network of externl fine hyphe in the soil 2. Dodd et l 3 reported tht the combintion tretments of AM fungi nd rock phosphte hve the potentil to increse plnt growth where phosphorus ws limiting plnt production. In soils with low P (phosphorus), AM fungi cn enhnce P uptke, plnt growth, nd root coloniztion 4. However, Jifon et l 5 reported tht inocultion with n AM fungus (Glomus intrrdices) depressed growth of Citrus urntium seedlings in soil with high P supply. Mny species of AM fungi hve worldwide distribution nd hve pprently dpted to diverse hbitts 6. There re mny tngerine (Citrus reticult) orchrds in northern Thilnd especilly in Fng District, north of Ching Mi. A preliminry survey reveled tht frmers pply fertilizers to their tngerine t different rtes, from very low to very high. This is likely to ffect benefits tht the trees my gin from ssocition with the AM fungi. In the mountins of northern Thilnd, we identified 29 species of AM fungi in 6 gener ssocited with shifting cultivtion 7. In Thilnd, however, informtion on AM ssocition with tree crops including tngerine is limited. This study therefore set out to investigte diversity nd bundnce of AM fungi in tngerine orchrds in Ching Mi province, northern Thilnd with different soil conditions, nd to evlute the effect of the indigenous AM fungi found in tngerine orchrds in Fng on growth of the ir lyered tngerine vriety Si Num Phung. MATERIALS AND METHODS Soil nd root smples from the study sites The study sites were tngerine orchrds in

2 260 ScienceAsi 34 (2008) Ching Mi province, northern Thilnd. Forty-five soil nd root smples (0 15 cm depth) were collected from the root zone of tngerine trees in 25 orchrds during the riny seson (June to September) in The soil ph of the study sites rnged from 4.4 to 7.5. Most soil smples in the orchrds were sndy cly lom nd only four tngerine orchrd soils were cly lom. The soil contined g/kg of totl nitrogen (Kjeldhl method), mg/kg vilble P (Bry II method), nd mg/kg extrctble K (1 N NH 4 OAc, ph 7). Evlution of rbusculr mycorrhizl coloniztion in tngerine root Root smples were wshed nd cut into 1 2 cm lengths. The root smples were clered in 10% KOH t 121 C for 15 min nd wshed in sieve under running wter. Clered roots were stined with 0.05% trypn blue in lctoglycerol t 121 C for 15 min. Thirty pieces of stined roots from ech smple were mounted on glss slides to evlute root coloniztion by AM fungi ccording to the method of McGonigle et l 8. Determintion of AM spores AM spores were seprted from 2 30 g of ech soil smple in the root zone of tngerine by wet sieving nd 50% sucrose centrifugtion 1. After centrifugtion, spores in the superntnt were poured over the 40 µm sieve nd wshed with wter to remove the sucrose before vcuum filtrtion on filter pper with gridlines. Spores on filter pper were kept in Petri dishes. Spores were counted under stereomicroscope. Different types of spores were selected to observe under compound microscope. Identifiction of AM fungi ws done ccording to morphologicl chrcteristics of published AM spore descriptions 9,10. Pot experiment for the effect of AM fungi on the host plnt The experiment ws full fctoril with AM fungl inocultion (inoculted nd un-inoculted tretments), two levels of N (ure t 0 nd 100 mg totl N/kg soil, N0 nd N100), nd two levels of P pplied (superphosphte t 0 nd 100 mg vilble P/kg soil, P0 nd P100) with four pots per tretment. The soil used in this experiment ws sndy cly lom, nd hd ph 6.0. The soil contined 0.41 g/kg totl N (Kjedhl method), 4.1 mg/kg vilble P (Bry II method), 44.0 mg/kg extrctble K (1 N NH 4 OAc, ph 7), nd 18.5 g/kg orgnic mtter (Wlkley-Blck method). Ech pot used in this experiment contined 15 kg sterile soil. Spores of AM fungi were isolted from indigenous soil of tngerine orchrds. About 150 spores of AM fungi were inoculted in the bottom of the plnting hole of inoculted tretments before plnting three feet long ir lyered cutting of Si Num Phung tngerine tree. The N nd P tretments were pplied in ten weekly doses. Ech pot received potssium chloride t the rte 50 mg K/kg soil by ppliction in ten weekly doses t the sme time s N nd P pplictions. The rtes of N, P, nd K ppliction were chosen to support moderte growth but not to suppress infection by the AM fungi. Ten months fter inocultion the lef, stem, nd root dry weights were determined. Roots from soil smples of inoculted tretments were used to determine root length coloniztion of AM fungi nd to determine spore densities in soil smples. Leves from ech of the tretments were evluted for N (Kjedhl method), P (dry shing nd molypdovndo-phosphoric cid), K (dry shing, nd tomic bsorption spectrophotometer method), nd Mg (dry shing nd tomic bsorption spectrophotometer method). Sttisticl nlysis Sttisticl tests were performed with SPSS version 10. The dt were nlysed by nlysis of vrince (ANOVA) to test the effect of the fctors. Men comprisons were determined by Wller- Duncn t p < RESULTS AND DISCUSSION Root coloniztion nd spore density of AM fungi ssocition with tngerine tree Soils in the region re generlly low in vilble P 11. Two thirds of smples contined > 250 mg P/kg indicting tht hevy rtes of P were pplied in the tngerine orchrds. The wide rnge of vilble P ws ssocited with vrition in root coloniztion nd rhizosphere spore density. Smples were grouped by cluster nlysis into 3 clsses by the level of vilble P: , , mg P/kg soil. Men percentge of AM coloniztion in the tngerine roots nd spore density in the rhizosphere for ech clss were found to be significntly different (p < 0.05). Root coloniztion declined in soil with higher vilble P. With more thn 500 mg P/kg soil, root coloniztion ws only hlf tht in soil with 250 mg P/kg or less (Fig. 1A). Spore density ws not significntly different in soil with mg P/kg, but ws depressed by 85% when vilble soil P exceeded 500 mg P/kg soil (Fig. 1B). Mny reserchers showed tht ppliction of high P levels suppressed root coloniztion of AM fungi in

3 ScienceAsi 34 (2008) 261 Root coloniztion (%) r = b b > > Avilble P (mg/kg soil) (A) Spore density (spores/100 g soil) r = b > > Avilble P (mg/kg soil) (B) Fig. 1 Mens of root coloniztion (A) nd spore density (B) of AM fungi in different levels of vilble P in soils from tngerine orchrds in Ching Mi province, northernthilnd during June to September Avilble P (mg/kg soil) (16 smples), > (14 smples), > (15 smples). Columns with different letters indicte significnt difference in the mens. Brs re + stndrd error of the mens. host plnts The results from this study showed much smller effect of P on root coloniztion by AM fungi thn those reported. For exmple, Nogueir nd Crdoso 14 reported tht root coloniztion of Gigspor rose nd Glomus intrrdices in soyben decresed shrply with incresing P levels from 50 to 200 mg/kg soil. The key to this difference my be the existence of diverse popultion of locl AM fungi tht respond differently to soil conditions. Mny reserchers reported tht there ws no reltionship between spore density nd root coloniztion Individul species of fungi produce spores ccording to their bility to proliferte in ech soil condition. Thus the mjor depression in spore density with >500 mg P/kg soil found in this study ws not mtched by ny depression in the percentge of root coloniztion. Diversity of AM fungi in tngerine orchrds A totl of 22 species of AM fungi ws found in the root zone of tngerine trees in ll soil smples (Tble 1). Bsed on morphology, they were plced in the four gener, Aculospor, Gigspor, Glomus, nd Scutellospor. All four gener were found in the three vilble P clsses of soil smples, but the species diversity nd number of species in ech genus differed between the soil P clsses. In the soil smples with mg/kg vilble P, 16 species of the AM fungi were found, in soil with more thn 250 mg/kg vilble P there were 12 or 13 species. Some species were found in ll three soil P clsses. Of the individul species, Glomus etunictum Becker & Gerdemnn nd Aculospor scrobicult Trppe were the most frequently found occurring in most smples. Some species ppered to be sensitive to high P. For exmple, A. morrowie, G. invermium, S. corlloide, nd S. nigr were only found in soil with 250 mg P/kg or less. Diversity of the fungi in ech genus lso showed some vrition with soil P. The number of species in Aculospor declined from four in the lowest P group to two in the highest P group. In Glomus nd Scutellospor, however, the number of species chnged only little with soil P, but the species themselves were different. In Scutellospor, the species found t 250 mg P/kg or less were entirely different from those found in soil with more thn 500 mg P/kg. These results suggest tht the AM fungi popultion depends on the vilble P. Bever 18 found tht AM fungl species, lthough ssociting with ll hosts, hve host-specific differences in their popultion growth rtes nd other Tble 1 Diversity of AM fungi with different levels of vilble P in soils from tngerine orchrds in Ching Mi province, northern Thilnd during June to September 2005.

4 262 ScienceAsi 34 (2008) components of the AM fungl community or other components of the soil community. A host vriety cn switch from comptible to incomptible mycorrhizl ssocitions with chnge in only one environmentl vrible 2. Effect of AM fungi on the host plnt in pot experiment Arbusculr mycorrhizl fungl inocultion incresed lef, root, nd totl dry weight of the ir lyered Si Num Phung tngerine tree (Tble 2). The effect of AM fungl inocultion on root dry weight of the host plnts disppered when the N nd P fertilizers were pplied together. Totl dry weights of inoculted plnts were significntly higher thn those in uninoculted plnts in pots with N but without P fertilizers. Hyphe of AM fungi ct s pump, supplying the root with supplement of wter nd minerl slts to which it normlly would not hve full ccess 19. This experiment found cler evidence of AM fungi influencing nutrition of the host plnt. Inocultion with AM fungi generlly incresed the concentrtion of N, P, K, nd Mg in tngerine leves (Tble 3). When the effect of AM on nutrient concentrtion ws combined with the effect on lef dry weight, the effect of AM on lef nutrient contents ws even more pronounced. Mny experiments reported tht AM fungi incresed the P contents of the host plnts. For other nutrients such s N, K, or Mg, AM fungi were reported both to hve nd not to hve n effect on the uptke these nutrients up to host plnts. It depended on the species of AM fungi nd the soil conditions In the pot experiment, root coloniztion by AM fungi nd rhizosphere spore density in tngerine lso showed response to N nd P tretments (Tble 4). Root coloniztion of the inoculted tretment ws mximum (42.9%) in the tretment N0P0, nd lowest (22.0%) in tretment of N100P100. Spore density ws lowest in tretments with P100, nd highest in N100P0. These vlues for 10 month old tngerine seedlings re much lower thn tht found on fully grown trees in the orchrds. Nevertheless, there were similr trends in which root coloniztion nd spore density of AM fungi were depressed by high P. In this experiment, 150 spores of mixed species of AM fungi were inoculted to ir lyered cuttings of Si Num Phung tngerine trees. The number of spores used for the inoculum my hve been too low to penetrte the corse roots of ir lyered cutting of tngerine. Clpperton nd Reid 24 Tble 2 Effect of AM fungl inocultion on lef, stem, root, nd totl dry weight (DW) of tngerine plnts in soils with different levels of N nd P, 10 months fter inocultion. M+, inoculted; M-, uninoculted with AM fungi; N0, N not dded; N100, 100 mg N/kg soil; P0, P not dded; P100, 100 mg P/kg soil with four pots per tretment. Mens in the sme column followed by different letters re significntly different. * = significnt; ns = not significnt.

5 ScienceAsi 34 (2008) 263 Tble 3 Effect of AM fungl inocultion on nutritionl levels in leves of tngerine in soils with different levels of N nd P, 10 months fter inocultion. CONCLUSIONS In spite of the generlly ccepted view tht root coloniztion by AM fungi is depressed by high P, this study hs found round 80% root coloniztion with up to 250 mg P/kg soil, nd only slightly less with up to 500 mg P/kg soil. While it ws confirmed in the pot experiment tht tngerine seedlings do benefit from AM fungi ssocition, it remins to be quntified how much the high degree of root coloniztion found in the field benefits the fully grown tngerine trees. The chnges in species of the AM fungi t different levels of vilble soil P suggest tht the diversity of locl AM fungi popultion should be closely exmined for better exploittion of this underground resource. ACKNOWLEDGEMENTS M+, inoculted; M-, un-inoculted with AM fungi; N0, N not dded; N100, 100 mg N/kg soil; P0, P not dded; P100, 100 mg P/kg soil with four pots per tretment. Mens in the sme column followed by different letters re significntly different. * = significnt; ns = not significnt. found tht there ws positive correltion between the proportion of inoculum dosge nd the mount of mycorrhizl coloniztion. Spore densities were very low in ll pot experiment tretments (Tble 4). The low densities of spores in this experiment my be due to the low percentge of root coloniztion of AM fungi. However, s mentioned bove, spore density nd root coloniztion re not necessrily closely correlted. Tble 4 Root coloniztion nd spore density of AM fungi in soils with different levels of N nd P (inoculted tretments). The uthors cknowledge finncil support from the Thilnd Reserch Fund nd the Commission on Higher Eduction. We re grteful to the Deprtment of Biology, Fculty of Science, Ching Mi University for the use of their fcilities. We lso thnk the Multiple Cropping Centre nd Knchnporn Lordkew for soil nlysis. REFERENCES 1. Brundrett M, Bougher N, Dell B, Grove T, Mljczuk N (1996) Working with mycorrhizs in forestry nd griculture. ACIAR Monogrph, Cnber. 2. Morton JB (1997) Yerbook of science nd technology. McGrw Hill. New York. 3. Douds DD, Schenck NC (1990) Cryopreservtion of spores of vesiculr-rbusculr mycorrhizl fungi. New Phytol 115, Grhm JH (2000) Assessing costs of rbusculr mycorrhizl symbiosis in groecosystems. In: Current dvnces in mycorrhize reserch (Podil GK, Douds DD, eds), pp APS Press, St. Pul, Minnesot, USA. 5. Jifon JL, Grhm JH, Drouillrd DL, Syvertsen JP (2002) Growth depression of mycorrhizl Citrus seedlings grown t high phosphorus supply is mitigted by elevted CO 2. New phytol 153, Abbott LK, Robson AD (1991) Field mngement of mycorrhizl fungi. In: The rhizosphere nd plnt growth (Keister DL, Cregn PB, eds), pp Kluwer Acdemic Publishers, Dordrecht, the Netherlnds. 7. Youpensuk S, Lumyong S, Dell B, Rerksem B

6 264 ScienceAsi 34 (2008) (2004) Arbusculr mycorrhizl fungi in the rhizosphere of Mcrng denticult Muell. Arg., nd their effect on the host plnt. Agrofor Syst 60, McGonigle TP, Evns DG, Miller MH (1990) Effect of degree of soil disturbnce on mycorrhizl coloniztion nd phosphorus bsorption by mizein growth chmber nd field experiment. New Phytol 116, Schenck NC, Perez Y (1988) Mnul for the Identifiction of VA Mycorrhizl Fungi 2nd edn, UNVAM Ginesville, Florid, USA. 10. INVAM website (2005) edu/fungi/ txonomy/clssifiction.htm. 11. Lnd Development Deprtment (2007) Chrcter nd qulity of soil: Ching Mi series. cm.htm (in Thi). 12. Khliq A, Snders FE (2000) Effects of vesiculrrbusculr mycorrhizl inocultion on the yield nd phosphorus uptke of field-grown brley. Soil Biol Biochem 32, Vlentine AJ, Osborne BA, Mitchell DT (2001) Interction between phosphorus supply nd totl nutrient vilbility on mycorrhizl coloniztion, growth nd photosynthesis of cucumber. Sci Hortic 88, Nogueir MA, Crdoso EJN (2007) Phosphorus vilbility chnges the internl nd externl endomycorrhizl coloniztion nd ffects symbiotic effectiveness. Sci Agric 64, Louis I, Lim G (1987) Spore density nd root coloniztion of vesiculr-rbusculr mycorrhizs in tropicl soil. Trns Br Mycol Soc 88, Smith SE, Red DJ (1997) Mycorrhizl Symbiosis 2nd edn, Acdemic Press, London. 17. Youpensuk S, Lordkew S, Rerksem B (2006) Compring the effect of rbusculr mycorrhizl fungi on uplnd rice nd Mcrng denticult in soil with different level of cidity. ScienceAsi 32, Bever JD (2002) Host-specificity of AM fungl popultion growth rtes cn generte feedbck on plnt growth. Plnt Soil 244, Dlpe P (1997) Biodiversity of mycorrhizl fungi. sols.htm. 20. Frey B, Schuepp H (1993) Acquisition of nitrogen by externl hyphe of rbusculr mycorrhizl fungi ssocited with Ze mys L. New Phytol 124, Mrschner H, Dell B (1994) Nutrient uptke in mycorrhizl symbiosis. Plnt Soil 159, Rutto KL, Mizutni F, Kdoy K (2002) Effect of root-zone flooding on mycorrhizl nd nonmycorrhizl pech (Prunus persic Btsch) seedlings. Sci Hortic 94, Tylor J, Hrrier LA (2001) A comprison of development nd minerl nutrition of micropropgted Frgri x nnss cv. Elvir (strwberry) when colonised by nine species of rbusculr mycorrhizl fungi. Appl Soil Ecol 18, Clpperton MJ, Reid DM (1992) A reltionship between plnt growth nd incresing VA mycorrhizl inoculum density. New Phytol 120,