You are on page 1of 8

Effect of Microbial Inoculants on the Indigenous Actinobacterial Endophyte Population in the Roots of Wheat as Determined by Terminal Restriction Fragment

Length Polymorphism
Vanessa M. Conn and Christopher M. M. Franco Appl. Environ. Microbiol. 2004, 70(11):6407. DOI: 10.1128/AEM.70.11.6407-6413.2004.

Downloaded from http://aem.asm.org/ on April 28, 2012 by guest

Updated information and services can be found at: http://aem.asm.org/content/70/11/6407 These include:
REFERENCES

This article cites 29 articles, 10 of which can be accessed free at: http://aem.asm.org/content/70/11/6407#ref-list-1 Receive: RSS Feeds, eTOCs, free email alerts (when new articles cite this article), more»

CONTENT ALERTS

Information about commercial reprint orders: http://aem.asm.org/site/misc/reprints.xhtml To subscribe to to another ASM Journal go to: http://journals.asm.org/site/subscriptions/

) (9. as were 28 to 42 indigenous actinobacterial genera present in the inoculated and uninoculated plants.asm. The results indicate that the addition of a nonadapted microbial inoculum to the soil disrupted the natural actinobacterial endophyte population. The most efficient nitrogen-fixing strains belong to the genera Rhizobium. sweet corn (Zea mays L. and citrus plants (1. Phone: 61-8-8204-5764. A portion of these microorganisms possess the ability to suppress disease and/or promote growth and are termed plant growth-promoting rhizobacteria (PGPR).) (17).) (19). specifically the genus Streptomyces (6. Vol. In order to achieve an increase in agricultural productivity in a sustainable manner. oilseed rape (Brassica napus L. PGPR were first used to improve crop fertility by increasing the amount of nitrogen available to the plant. A number of the biologically active endophytes and rootcolonizing microorganisms that have been isolated or detected belong to the actinobacterial phylum. 2). and Nocardioides albus EN46 or from untreated seeds sown in soil with and without a commercial mixed microbial soil inoculant. Bradyrhizobium.1128/AEM. 29). Bedford Park. All Rights Reserved.) (8.6407–6413. and Allorhizobium (3). Bedford Park. Flinders University. Sturz et al.. 19). The endophytic actinobacterial population within the roots of 6-week-old wheat plants was assessed by T-RFLP. There appears to be a close relationship between the rhizosphere and endophyte populations. * Corresponding author. sepedonicus. Australia Received 9 March 2004/Accepted 29 June 2004 The effect of single actinobacterial endophyte seed inoculants and a mixed microbial soil inoculant on the indigenous endophytic actinobacterial population in wheat roots was investigated by using the molecular technique terminal restriction fragment length polymorphism (T-RFLP).org/ on April 28. wheat (Triticum aestivum L. A major factor influencing plant growth and health is the microbial population living both in the rhizosphere and as endophytes within healthy plant tissue.11. Recently. strain EN27. Downloaded from http://aem. cotton (Gossypium hirsutum L.Franco@flinders.) (6.au. Franco* Department of Medical Biotechnology. 12.70. Streptomyces sp. Rhizoctonia solani. School of Medicine. p. and Verticillium spp. For example. 2012 by guest Cereal production is an important source of food grains and is expected to play a key role in meeting the needs of the world population. No. Flinders University. 6407 Endophytic microorganisms may offer an advantage in terms of efficiency of colonization leading to improved effectiveness. 26. when complementary crops were grown in rotation they shared 70% of the same species of endophytic bacteria (25). This was in contrast to the addition of a single actinobacterial endophyte to the wheat plant. Fax: 61-88204-4101. Microbial biocontrol agents have been shown to inhibit soil-borne pathogens such as Fusarium oxysporum. M. reducing diversity and colonization levels. it was shown that a Streptomyces lydicus strain previously isolated from rhizosphere soil could colonize root surfaces and cause physiological changes to the nodules of pea plants (26). 18. 23. Sinorhizobium. Colonization of the wheat roots by the inoculated actinobacterial endophytes was detected by T-RFLP. belonging to the genus Frankia. Mailing address: Department of Medical Biotechnology. 14. Mesorhizobium. Gaeumannomyces graminis.) (17). 22). there will be increased reliance on manipulation of microorganisms that are beneficial to soil and plant health. 6407–6413 0099-2240/04/$08. As demonstrated in some cases. Phytophthora spp. is a nitrogen-fixing actinobacterium that forms actinorhizae with eight . Australia. strain EN2. South Australia 5042. 29. In oak. Wheat was cultivated either from seeds coated with the spores of single pure actinobacterial endophytes of Microbispora sp. Nov. potato (Solanum tuberosum L. Azorhizobium. where the increase in colonization level could be confirmed even though the indigenous endophyte population was not adversely affected. 30).00 0 DOI: 10. 70. which is growing by 160 persons per min (11).2004 Copyright © 2004. The presence of the commercial mixed inoculant in the soil reduced the endophytic actinobacterial diversity from 40 genera to 21 genera and reduced the detectable root colonization by approximately half. The first actinobacterial endophyte isolated.APPLIED AND ENVIRONMENTAL MICROBIOLOGY. 11 Effect of Microbial Inoculants on the Indigenous Actinobacterial Endophyte Population in the Roots of Wheat as Determined by Terminal Restriction Fragment Length Polymorphism Vanessa M. Some success has been achieved in controlling crop pathogens and promoting plant growth by supplementing the crop soil with these PGPR and other biocontrol microbial inocula (3). Conn and Christopher M. Endophytic bacteria have been isolated from a variety of plants. Endophytic bacteria have the ability to promote growth and inhibit plant disease. South Australia. 22. 9. 21. However.edu. 2004. (24) found that 61 of 192 endophytic bacterial isolates from potato stem tissues were effective biocontrol agents against Clavibacter michiganensis subsp. Pythium spp. and as they are in intimate contact with the plant they are an attractive choice as biological control agents. American Society for Microbiology. endophytic bacteria biologically active against the oak wilt pathogen Ceratocystis fagacearum have been isolated (4). 24). a large number of biocontrol agents fail to be effective due to the difficulty of manipulating the highly complex rhizosphere environment. E-mail: Chris. including tomato (Solanum lycopersicum L. 8. (7..

One-hundred liters of this microbial suspension was added to soil at a rate of 100 liters per hectare. and N. All plants were cultivated in a glasshouse with watering as required. Data was analyzed using the GeneScan Analysis program V. The pure cultures of the three actinobacterial endophytes. with the major genera being Streptomyces. Microbispora sp. and Nocardioides albus EN46.. Statistical and data analysis. Plates were incubated at 27°C for 3 to 10 days until abundant sporulation had occurred. Investigation of the effect of a mixed microbial soil inoculant was performed by cultivating wheat seeds (cv. Actinobacterial endophytes were isolated from healthy wheat roots previously (6).6 419.4 240. 232–235 470–472. MATERIALS AND METHODS Actinobacterial cultures. Endophytic bacterial DNA was extracted from the roots of 6-week-old wheat plants. resulting in six replicate PCR products for each treatment. Streptomyces sp.asm. Streptomyces sp. The petri dish was left in the laminar flow cabinet overnight to evaporate the water and coat the spores onto the seeds. Actinobacterial cultures of Streptomyces sp. The minimum and maximum abundance percentages were calculated by dividing the area of the peak of interest by the total peak areas for that particular T-RFLP profile. and MboI (Promega). It is well known that the addition of nonindigenous microbes to soil can affect the indigenous rhizosphere population (28). Table 1 shows the fragment sizes obtained from the digestion of the 16S rRNA gene sequences from each endophyte used in this study and for each restriction enzyme. The soil was collected for immediate use in pot trials. located on the Eyre Peninsula.au) for 24 h before use. T-RFLP analysis of 16S rRNA gene amplification products. indicating their potential use as biocontrol agents (7). or half-strength potato dextrose agar (PDA). oatmeal agar (OA). albus EN46 were grown on MS agar and PDA. Krichauff) in soil obtained from Swedes Flat. strain EN27. Two milliliters of the actinobacterial spore suspension was poured over the seeds and mixed. The terminal restriction fragment (TRF) sizes present for each restriction enzyme were determined from the GeneScan data. strain EN27. Adelaide. using 1 l of the restriction digest. including Rhizoctonia solani. strain EN2. Microbispora.7 -tetrachlorofluorescein)-labeled 1492r (5 TA CGG GTA CCT TGT TAC GAC TT 3 ) (27) prepared by GeneWorks.nutri-tech. However. strain EN2. The root material was first surface sterilized by using an ethanol-sodium hypochlorite procedure. and MboI. MICROBIOL. Streptomyces sp. available as granules. A representative T-RFLP profile obtained with HinfI for one of these plant treatments is shown in Fig. EN27. and the endophytic bacterial DNA was extracted as described previously (5). EN27. The size of the terminal 16S rRNA gene fragments present in the restriction digestions were determined on an automated Applied Biosystems 373 DNA sequencer.2 (Applied Biosystems). Table 2 shows the three specific TRFs corresponding to the inoculated endophytes and the minimum and maximum abundance percentages of the peak areas (not corrected). HhaI. DNA extraction from actinobacterial cells. Seeds coated with the actinobacterial spores were cultivated in pots (100 mm height. 1. and four fungal species (NutriTech. Total genomic DNA was extracted from the actinobacterial spores by using a modified cetyltrimethylammonium bromide/NaCl protocol (13) as described previously (5). cellulosae. EN2. Stretch. using 10 l of the PCR mixture for 16 to 18 h to achieve complete digestion. Cultures were maintained on mannitol soy (MS) agar.com.7 162. Nutrilife 4/20. Coating wheat seeds with actinobacterial spore inoculum. albus EN46. personal communication).1. one of which was treated with the commercial inoculant (NutriLife 4/20.4. Australia.5 162. and Franco (5). and N. albus EN46. strain EN27. and then the digests were stored at 20°C. There were unique fragment sizes for each of the added endophytes and restriction enzyme. Micromonospora. EN46. the effect of microbial inoculants on the endophytic population is unknown. HhaI. Spores were harvested from the plate with a sterile loop and were suspended in 2 ml of sterile water. NutriTech). Each suspension contained approximately 1012 spores/ml. located in the southeast of South Australia. Microbispora sp. NutriLife 4/20 (NutriTech. Growth of actinobacteria and harvesting of spores. 50 mm diameter) containing soil obtained from a wheat field in Haslam.3 418.3 178. In this study the effect of a commercial mixed microbial inoculant. HhaI. and Nocardioides (6). Plants were harvested 6 weeks after germination. and MboI Species (GenBank accession no. Semiquantitative analysis of the T-RFLP data was carried out as described in Conn and Franco (5). 215–218 236–240 419 158 175 410 157 175. Partial 16S rRNA gene sequences were amplified from the endophytic DNA using the actinobacteria-biased primers 243f (5 GGA TGA GCC CGC CGC CTA 3 ) (10) and 5 TET (6-carboxy-2 . Untreated seed was cultivated and used as a control sample. 15) was used in conjunction with a semiquantitative analysis protocol (5). Streptomyces sp. strain EN2. 490 162. For all plant treatments three seeds were sown per pot and three replicate pots were sown. The granules were mixed with the NutriTech nutrient formulation in water according to the manufacturer’s instructions (www.org/ on April 28. Three replicate root samples were used for each treatment.7. The culture-independent technique terminal restriction fragment length polymorphism (T-RFLP) (10. albus EN46 16S rRNA gene sequence digested with HinfI. A number of endophytic actinobacteria were previously isolated by culture-dependent methods. Pythium spp. Approximately 40 wheat seeds (cultivar Krichauff) were placed in a sterile petri dish. Wheat cultivation. A number of these isolates were capable of suppressing fungal pathogens of wheat in vitro and in planta. respectively.5 410. investigated in the study were analyzed by the T-RFLP technique to determine the unique TRFs obtained with the restriction enzymes HinfI. and Gaeumannomyces graminis var tritici. contained 20 bacterial strains.7 163.7 Downloaded from http://aem. The . RESULTS Detection of introduced endophytes in the roots of wheat by T-RFLP. including Streptomyces albidoflavus and S.3. TABLE 1. Two amplifications per sample were carried out according to Conn EN2 (AY148073) HinfI HhaI MboI EN27 (AY14805) HinfI HhaI MboI EN46 (AY148081) HinfI HhaI MboI 179. and single actinobacterial endophytic inoculants on the indigenous actinobacterial endophyte population of wheat was investigated. South Australia. Two soil samples were obtained from this site. the annotated peaks indicate the fragment corresponding to the introduced actinobacterial endophyte. Fragment sizes of Microbispora sp. Eumundi. Extraction of endophytic bacterial DNA from the roots of wheat. N.6408 CONN AND FRANCO APPL. The endophytic bacterial DNA isolated from the roots of endophyte-inoculated and uninoculated wheat plants at 6 weeks of growth was subjected to the T-RFLP procedure. Single restriction digestions of each of the 16S rRNA PCR products were performed with HinfI. 2012 by guest a EN2. Australia). whereas Microbispora sp.) and enzymea Predicted fragment size (bp) Actual fragment size (bp) families of angiosperms (20). ENVIRON. strain EN27 and N. Actinobacterial cultures used in this study were Microbispora sp. which enabled the introduced endophytes to be detected in the roots of wheat by corresponding the fragment sizes and peak areas to the specific inoculant. strain EN2 was grown on OA.

albus EN46. Streptomyces sp.61 3.58 1. 2012 by guest a EN2. but in the presence of added NutriLife 4/20 inoculum the number of detectable genera was reduced to 21. Analysis of the specific fragments for N. The highlighted peaks correspond to the specific fragment of the specific actinobacterial endophyte inoculated onto the seed.26 2. 16). These fragments were corresponded to bacterial species by using the TAP T-RFLP database (16). EN27-inoculated seed (c). HhaI. Figure 2 shows the GeneScan T-RFLP profile of the actinobacterial 16S rRNA gene TRFs amplified from the roots of wheat grown in inoculated and uninoculated soil and digested with HinfI. while the MboI-specific fragment increased 2. strain EN27. percentages were calculated for each replicate in the plant group.96 10. and fertilizers with microbial inoculants due to environmental and health concerns.96 2. Streptomyces sp.87 6. N. It is well known that the addition of nonindigenous microbes to the soil can affect the Downloaded from http://aem. respectively. The actinobacterial 16S rRNA gene was amplified from the endophytic bacterial DNA extracted from the roots of wheat grown from inoculated or uninoculated seeds. compared to that of plants grown in the uninoculated soil. The specific 241-bp HinfI fragment corresponding to Streptomyces sp. 21).org/ on April 28.69 3. a total of 58 genera were identified. EN2-inoculated seed (b).VOL.58 1. Among the four different plant treatments. HhaI.98 1. The actinobacterial 16S rRNA gene terminal fragment sizes obtained with HinfI.62 0.45 23.13 5.02 1.33 3.57 10. DISCUSSION There is an emerging trend to replace chemical herbicides.1-fold.94 11. the effect of these inoculants on the indigenous endophytic actinobacterial population was determined by T-RFLP and was compared to that of the uninoculated control. strain EN2. and N. ND. indicating that colonization has occurred. and MboI are shown in Table 3. a number of PGPR are used as biological control agents for the suppression of soil-borne diseases (1. The effect of the commercial mixed microbial inoculant on the endophytic actinobacterial population in the roots of wheat was determined by T-RFLP analysis of the 16S rRNA gene sequence. 1.2-fold in terms of maximum abundance percentage. EN27. Microbispora sp. In addition. In 2001. albus EN46-inoculated seed (d).08 3. 70. not determined. albus EN46 showed that the HhaI and HinfI peaks decreased.33 10. These triple TRFs were corresponded to bacterial species by using the TAP-T-RFLP database (5. strain EN27 was not present in the uninoculated control.6% in all treatments are listed in Table 4. Forty actinobacterial genera were detected in the untreated soil.41 9. The level of relative colonization was also lowered. Microbispora sp. Thirty-six genera were present in all treatments.asm. between a range of 14 to 86%.62 ND 1. The maximum abundance percentage of each actinobacterial genus present and the percentage of reduction of genera in the inoculated soil are shown in Table 6.19 0. From these results it was observed that the maximum abundance percentage for the MboI fragment of Microbispora sp.69 3.38 4. As the TRFLP technique was able to detect colonization of the introduced endophytes. The TRF sizes obtained in at least four of the six replicates with the restriction enzymes HinfI. TABLE 2. and 45 genera were present in three of the four treatments. fungicides.69 1. 2004 EFFECT OF MICROBES ON WHEAT ROOTS AS DETERMINED BY T-RFLP 6409 FIG. The effect of a commercial mixed microbial soil inoculant on the endophytic actinobacterial population in the roots of wheat.62 3.17 10. Comparison of the minimum and maximum fragment percentages obtained from T-RELP profiles of endophytic bacterial DNA isolated from the roots of wheat inoculated with an endophyte to those of an uninoculated plant (n 6)a Fragment % for inoculated wheat Min Max Fragment % for uninoculated wheat Min Max Restriction enzyme and inoculant Fragment of interest (bp) EN2 HinfI HhaI MboI EN27 HinfI HhaI MboI EN46 HinfI HhaI MboI 176 420 162 241 420 163 179 411 162 0.13 strain EN2 increased by 1.19 0.06 5. as the peak areas are specific for each T-RFLP profile. 65% of the nitrogen supply to crops originated from PGPR. and the HhaI-specific fragment increased by twofold in terms of maximum abundance percentage. Effect of introducing actinobacterial endophytes on the indigenous endophytic population of wheat roots. and MboI that were present in at least four of the six replicates are shown in Table 5. This provided a good indication that colonization had occurred.77 4. whereas the HinfI and HhaI fragments increased by approximately two. T-RFLP HinfI profiles for the roots of wheat grown from uninoculated seed (a).80 7.78 14.55 5.13 ND 2.and threefold. . The 49 genera which had a maximum abundance percentage of more than 0. EN46. The minimum and maximum abundance percentages were determined for each specific TRF from the six replicates.

30 0.14 0.56 0.38 0.86 4. albus EN46 appeared to Kribbella Streptomyces Bifidobacterium Arthrobacter Nocardia Actinoplanes Rhodococcus Kineococcus-like bacterium Nocardioides Rubrobacter Geodermatophilus Mycobacterium Microbispora Brevibacterium Thermomonospora Saccharomonospora Corynebacterium Frankia Kitasatospora Microbacterium Actinosynnema Spirilliplanes Streptoalloteichus Agromyces Catellatospora Cellulomonas Gordonia Lechevalieria Lentzea Micrococcus Pimelobacter Sarraceniospora Terrabacter Actinomyces Saccharothrix Thermocrispum Aeromicrobium Candidatus Microthrix Cellulosimicrobium Pilimelia Prauseria Promicromonospora Actinobaculum Actinomyces Parvopolyspora Planobispora Sanguibacter Streptomonospora Williamsia 13.04 3.08 1.32 0.28 1. A previous study by Coombs and Franco (7) showed these isolates were all capable of significant suppression of disease symptoms caused by G.08 1.62 1.10 1.27 2.43 0.6% in all samples were omitted) (n 6) Genus Uninoculated EN2 EN27 EN46 TABLE 3.14 0.53 2. The T-RFLP technique was first assessed for the ability to detect a single actinobacterial endophyte introduced to the roots of wheat by seed-coating application.46 1.43 1.14 0.18 0.27 1.23 1.64 0.26 4.91 0.37 1.00 6.71 0.41 1.54 2.05 5.54 0.27 2.00 0. as the specific peak areas corresponding to this strain decreased for the HinfI and HhaI fragments and increased significantly for the MboI fragment for one of the replicates.76 0.28 0.73 0.64 1.14 0.28 0.08 1.27 1.25 0. Maximum abundance percentage of actinobacterial genera in the roots of wheat grown from seed inoculated with endophytes (Microbispora sp.00 0.64 0.92 2.00 0. albus EN46 [46]) or without (UN) an endophyte and grown for 6 weeks in soil obtained from Haslam (n 6) HinfI UN 2 27 46 HhaI UN 2 27 46 MboI UN 2 27 46 34 39 45 49 53 58 62 63 67 70 76 81 84 88 91 95 97 100 112 126 132 147 163 167 176 178 180 189 201 214 219 232 237 241 348 354 369 34 39 42 45 49 53 58 63 70 74 77 81 83 88 91 95 97 100 106 120 112 126 132 147 162 175 190 200 214 227 279 332 348 369 387 398 412 413 416 420 422 470 34 36 39 45 49 53 58 61 64 67 70 77 81 82 84 88 92 95 97 100 112 132 134 153 158 162 163 166 173 175 183 214 218 284 287 indigenous ectorhizosphere population.28 0. EN27 [EN27].30 0.00 0.42 4. and N. strain EN27 also .37 1.14 0. strain EN27.28 1.10 0.37 1.18 1.66 15.00 2.65 1.98 0.6410 CONN AND FRANCO APPL. and N. The specific TRF peaks corresponding to Microbispora sp.14 7.00 13. TABLE 4.64 1.37 11. HhaI.08 0.37 1. Streptomyces sp. strain EN27 colonized the wheat root from the increase in peak area corresponding to the specific fragments. Streptomyces sp.14 0.43 0. strain EN2 and Streptomyces sp. strain EN27.54 1.00 0. N.92 0.00 0. albus EN46 [EN46]) and uninoculated seed for 6 weeks in soil obtained from Haslam (genera with a maximum percent under 0.07 0.86 0.62 1. Streptomyces sp.00 0.68 20. ENVIRON. Streptomyces sp.00 0.32 Downloaded from http://aem.56 1.13 1.28 0.16 0.37 1.61 0.6 7.33 1.43 1.73 1.66 0.16 2.75 2.4 0. albus EN46 digested with HinfI. MICROBIOL.43 1.54 0.64 0.08 1.37 1.96 3.65 0.28 1.37 13.54 0.43 0.46 1.86 0. N. EN27 [27].00 1. graminis var tritici at 4 weeks.46 2.00 1.08 1.19 30.68 0.27 2.37 1.54 0.asm.10 0. Hha.28 0.00 0.00 1. EN2 [2].32 0.45 2.14 0. 16S rRNA gene sequence fragment sizes (in base pairs) obtained with the restriction enzymes HinfI. 2012 by guest occur only in one of the three replicate plants.28 0.01 3.org/ on April 28.07 2.72 12.00 0.14 0. The endophytic actinobacteria Microbispora sp.86 0.62 1.86 0.32 1.37 1.24 0.38 4.54 0.65 0.87 0.02 3.09 0.03 3.08 1.27 1.26 2.14 0.63 0.04 0.93 3.08 1.79 3.39 0.00 0.08 1.53 0.08 0.92 5. strain EN2. EN2 [EN2].64 0.14 0.76 0.54 0. strain EN2.18 2.32 0. and MboI were used to monitor colonization levels in wheat roots.34 4.21 18.15 6. and MboI for the roots of wheat inoculated with (Microbispora sp.62 1.37 1.2 3.00 3.83 0.53 0. The results indicated that Microbispora sp.14 0.81 2.98 2.08 1.00 1. but the effect of microbial inoculants on the endophyte population was unknown.43 0.12 3.66 1.08 4.34 0. Colonization of N.86 0.64 0.46 1. albus EN46 were all previously isolated from wheat roots (6).31 1.32 0.04 0.29 3.38 0. Streptomyces sp.37 1.37 1. This study used T-RFLP analysis to assess the effect of a mixed microbial soil inoculant and single endophytic actinobacterial seed inoculants on the indigenous endophytic actinobacterial population in the roots of wheat.37 1.30 0.14 0.39 0.

2012 by guest TABLE 5. The T-RFLP results have shown that at 6 weeks the introduced endophytes increase in colonization level by up to threefold.6%. 70. and MboI for the roots of wheat grown for 6 weeks in field soils obtained from Swedes Flat with and without NutriLife 4/20 (n 6) HinfI Without NutriLife 4/20 With NutriLife 4/20 HhaI Without NutriLife 4/20 With NutriLife 4/20 MboI Without NutriLife 4/20 With NutriLife 4/20 35 39 41 44 49 53 57 61 63 70 80 82 84 89 92 94 97 99 105 112 128 133 147 167 174 176 178 180 188 213 236 258 279 284 321 348 369 407 411 36 39 41 44 49 53 58 61 63 70 81 85 89 91 94 97 99 106 113 120 127 133 147 175 178 190 213 227 239 258 279 321 348 368 372 385 387 410 412 414 416 418 420 34 39 43 49 53 57 63 70 72 80 82 89 91 95 99 105 113 118 128 133 147 156 158 162 166 173 175 178 214 258 . 2004 EFFECT OF MICROBES ON WHEAT ROOTS AS DETERMINED BY T-RFLP 6411 FIG.asm. suggesting that the introduced endophytes do not need to colonize at high levels to exhibit a positive effect on the wheat plant.org/ on April 28.VOL. 2. with approximately 92% of the genera detected present in at least three of the treatments. As the T-RFLP technique was able to detect the introduced endophytes. The T-RFLP technique detected 58 actinobacterial genera. The predominant genera in all treat- Downloaded from http://aem. HhaI. This is of importance in relation to the use of endophytes as biological control agents. showed significant growth regulation of wheat at 4 weeks. T-RFLP profile for the roots of wheat grown for 6 weeks in field soil from Swedes Flat without NutriLife 4/20 (a) and with NutriLife 4/20 (b) and digested with HinfI. the technique was used again to determine whether the indigenous endophytic actinobacterial population changes in response to the introduction of a single actinobacterial endophyte. 16S rRNA gene sequence TRFs (sizes in base pairs) obtained with HinfI. with 49 genera having a maximum abundance percentage of more than 0. The results indicated that the introduction of an endophyte to wheat roots did not disrupt the indigenous endophytic actinobacterial microflora.

However.69%.25 6.00 100.00 16.00 2.00 1380.39 0.00 0.06 1.76 9. and P. W.00 0.49 9.05 5.02 50. van Vuurde. C.00 6.00 0.00 53.00 100.53 to 9.98 7.06 1.08 0. REFERENCES 1. both the endophytic actinobacterial population and the relative level of colonization were reduced by approximately half. The T-RFLP method detected 40 actinobacterial genera present in the roots of wheat grown in Swedes Flat soil without the added microbial inoculant. Microbiol. L. Kribbella was detected in the noninoculated control at 13.02 20.00 100.65 100. increased from 1.51 2. These actinobacterial genera were not components of the inoculant. Lugtenberg.05 51. albidoflavus have not been reported to have an endophytic association with plants.08 2.79 100. L.68% in the Streptomyces sp.46 4.00 0.02 100. and S.19%.39%.06 to 2. The conclusions from this study are that reintroduction of an endophyte to a wheat seed does not significantly affect the indigenous endophytic actinobacterial population. Streptomyces.. Bifidobacterium. W.72 4. which in turn may have a negative effect on plant growth and disease resistance.15%. albus EN46-treated (30.72 and 13. Microbiol.org/ on April 28.17 0.66%) roots. I. ACKNOWLEDGMENT This work was supported by the Australian Grains Research and Development Corporation (GRDC). A.99 4. J.00 2. indicating that indigenous actinobacterial endophytes have a role in maintaining plant health. Can.82 9. This is in contrast to the results obtained when a mixed microbial inoculant was added to the soil in which the wheat was cultivated. TABLE 6.08 1.42 2.00 100.82 21. 3. Aguilar-Vidoso.95 2. Kribbella species decreased to 7. There were three genera for which the maximum abundance percentages increased in soil with the added inoculant. decreased from 18. Arthrobacter. Marcon.62 80. ENVIRON. The presence of the mixed commercial inoculant appears to prevent a number of actinobacterial genera from access to the germinating seed.00 39. Appl. Nocardia.06 to 15. Many biocontrol strains are capable of improving plant growth and disease resistance in controlled pot trials but have been unsuccessful when taken out into the field. a number were capable of causing significant disease resistance and growth promotion (7). Opin.47 4.00 0.19 100.82 0.15% (noninoculated) to 20.59 1. albus EN46-treated plant.. strain EN27inoculated plants.01 0. and Nocardioides spp.75 2.00 0. albidoflavus and S.00 1.08 2. Thermomonospora spp.64 18. When NutriLife 4/20 was added.49 100. J.00 100. Variability and interactions between endophytic bacteria and fungi isolated from leaf tissues of citrus rootstocks.00 100. 2012 by guest . cellulosae. Maccheroni. D. These results suggest the microbes present in the NutriLife 4/20 inoculum are able to outcompete the indigenous actinobacterial microflora.44 6.53 16. 2001. the Streptomyces species also increased in the Microbispora sp. J.46 3. strain EN27-inoculated plants. Araujo. strain EN2-inoculated plants and at 13. EN2 (18. in the N. However. The Streptomyces species increased from 12. G.00 0.00 53. and the relative level of colonization of individual genera decreased to a range of 14 to 86%.08 0. and B. Molecular basis of plant growth promotion and biocontrol by rhizobacteria. 2002. Not all soil microbes are expected to have the capability of endophytic colonization. hence their presence may be site specific or they may posses a higher level of colonization competitiveness compared to that of other indigenous microflora.00 15.60 54.16 0. van Elsas.69 0.24 0. with the exception of Kribbella.00 100.43 54.04 100. Azevedo.84 4.. In contrast.65 51.41 7.99 4.00 ments were Kribbella. cellulosae and S.00 100. the addition of a mixed microbial inoculant to the soil reduces the endophytic actinobacterial diversity and relative level of colonization.82%. W.88 6. V.37%). leading to a reduction of endophytic colonization of the wheat roots.65 100.48 2.00 57.07 2. and Thermomonospora. Even though the mixed inoculant contained S.42 2. This knowledge is of importance because endophytes have a role in maintaining plant health.00 58. Downloaded from http://aem. Nevertheless.00 51. Environ.47 to 6. J.04 6. and Rhodococcus.42 2.12% in the Streptomyces sp. the results indicated the endophytic actinobacterial population was relatively stable despite the inoculation of an endophyte.52 7.06 1. The actinobacterial genera detected was reduced to 21 genera.00 2. Maximum abundance percentage for each genus identified by T-RFLP in the roots of wheat grown in soil obtained from Swedes Flat with and without NutriLife 4/20 (n 6) % With NutriLife 4/20 % Without NutriLife 4/20 Decrease (%) of genera in soil with NutriLife 4/20 Genus Mycobacterium Bifidobacterium Rhodococcus Streptomyces Nocardia Geodermatophilus Saccharomonospora Arthrobacter Microbacterium Frankia Gordonia Saccharothrix Brevibacterium Thermonospora Kitasatospora Pimelobacter Lentzea Agromyces Actinomyces Williamsia Thermocrispum Sanguibacter Rubrobacter Corynebacterium Micrococcus Micromonospora Leifsonia Dietzia Curtobacterium Brachybacterium Kineococcus-like bacterium Actinosynnema Actinoplanes Catellatospora Sarraceniospora Nocardioides Lechevalieria Kribella Streptoalloteichus Spirilliplanes Planomonospora 9. and J. Diversity of endophytic bacterial populations and their interaction with Xylella fastidiosa in citrus plants. Nocardioides.82 100.17 1.75 13. the abundance percentage of endophytic Streptomyces spp.asm.53 100.24 0. L. Curr.00 0.21% in Microbispora sp. 2001. L.6412 CONN AND FRANCO APPL.00 166. Plant Biol. 2.23 29.00 1. W.00 100.99 4.00 100. Barroso. MICROBIOL. V.15 3. W.46 4.and N. 47:229–236.00 0.46 4. 68:4906–4914.00 2.00 0.32 100. 4:343–350. increased from 5.62 86.78 20.00 100.00 2.08 0.36 40.00 4.06 1.65 14.00 6.01 1.17 2. Maccheroni. Araujo.00 2. It is possible that the addition of biocontrol strains to the soil causes disruption of the natural endophyte population.00 15. This has been attributed mainly to the inability to compete in the complex rhizosphere environment. J. Kribbella species increased from 1.42 2. Bloemberg. Of the endophytic actinobacteria isolated from the wheat roots by Coombs and Franco (6).

P. and D. Lane. G. W. and B. and N. and J. G. H. J. 28:27–33. Appel. de Freitas. Novel plant-microbe rhizosphere interaction involving Streptomyces lydicus WYEC108 and the pea plant (Pisum sativum). Microbial interactions and biocontrol in the rhizosphere.. 41:895–901. 173:697– 703. L. Wellington. England. A.. A. 27. Bibb. Deobald. 2002. D. 14. and C. Coombs. Appl. Garbeva. Paulitz. S. Microbiol. Huci. and C. Conn. Microbiol.. F. S. Plant Pathol. T. Microbiol.. P. Benhamou. T. and J. J. Biocontrol within the context of soil microbial communities: a substrate-dependent phenomenon. Differences in the microbial communities associated with the roots of different cultivars of canola and wheat. Biol. L. 70:1787–1794. Parzy. and J. S.. 41:369–383. 26. Plant Pathol.. Salove. John Innes Centre. J. and P. W.. J. 18. D. M. Crawford. M.. A. Saxman. Environ. B. Analysis of actinomycete communities by specific amplification of genes encoding 16S rRNA and gel electrophoretic separation in denaturing gradients. J. 2002. 37:427–446. and M. Practical Streptomyces genetics.. Reeves.. E. 50:301–320. 1998. Barns.. N. A. Associations of bacterial endophyte populations from red clover and potato crops with potential for beneficial allelopathy. Gonzalez. A.). M. Exp. P. Analysis of the endophytic actinobacterial population in the roots of wheat (Triticum aestivum L. V. N. A. Matheson. Appl. K. R. 44:162–167. 2001. T. D. J. 2000. M. Microb. A. tritici in wheat. M. Jung. Phytopathol. 61:3119–3128. J. Kaiser.) by terminal restriction fragment length polymorphism (T-RFLP) and sequencing of 16S rRNA clones. Hoitink. D. and N. Heuer. Ecol. K. Can. Buttner. Kloepper. Natl. F. H. Tiedje. Plant genetic resources: what can they contribute toward increased crop productivity? Proc. K. 5. Kloepper. Downloaded from http://aem. Isolation and identification of actinobacteria isolated from surface-sterilized wheat roots. Theor. J.. W. Zehnder. J. Can. A. Sturz. Sikora. Bot. R. B. Environ. 70. D. Skovmand. S. Biol. Reiter. M. U. Biol. 2003. R. 6. 1998. Ribaut. A. Sci. Matheson. 67:4742– 4751. L. N. 23. Strap. 20.).) and wheat (Triticum aestivum L. Control 4:373–381. Buchanan. Biol. W. 1997.. J. J. M’Piga. Analysis of endophytic bacterial communities of potato by plating and denaturing gradient gel electrophoresis (DGGE) of 16S rDNA based PCR fragments. P. 2002. 48:360–369. J. J. M. M. 22. and A. 19. 8. C. M. 13. Yuan. T. A. 66:3616–3620. A. Control 18:208–215. Taba. S. Pelletier. Appl. 28. Nejad. W. and I. P. Crawford. FEMS Microbiol.. J. J. K. Acad. and M. Kieser. FEMS Microbiol. P. Annu. Can. 25. R. A. 1991. and E.. Sessitsch. Heuer. Plant Mol. and J. Franco. V. Zock... Provorov. Whipps. A. 1995. M. Evaluation of endophytic actinobacteria as antagonists of Gaeumannomyces graminis var. Rodriguez-Kabana. Pfeifer. van Elsas. Biological control of Phytophthora root rots on alfalfa and soybean with Streptomyces. Microbiol. and G. 2001. Samac. Endophytic bacterial communities in the periderm of potato tubers and their potential to improve resistance to soil-borne plant pathogens. 9. Appl.. T. P. Buchner. Borisov.. Use of the T-RFLP technique to assess spatial and temporal changes in the bacterial community structure within an agricultural soil planted with transgenic and non-transgenic potato plants. G. Microbiol. Murphy. G. M. F. Baker. Endophytic bacteria induce growth promotion and wilt disease suppression in oilseed rape and tomato. 2012 by guest . Smalla. F. Appl. 1997. 21. sp. H. radicis-lycopersici in tomato plants treated with the endophytic bacterium Pseudomonas fluorescens strain 63–28. Boehm. 2000. Microbiol.. Wieland.. Cole. J. Krsek. 44:844–851. J. Control 23:285–295. M. Environ. Microbiol. Germida. and M. Michelsen. R. Xaio. L. and T. J. J. J. Environ. M. Environ. C. Franco. B. Biol.. T. Johnson. L. 2001. Plant root-bacterial interactions in biological control of soilborne disease and potential extension to systemic and foliar disease. 1999. 1305:1–10. T. S. Norwich. B.. J. Franco. and E. Belanger. 1999. van Vuurde. Evaluation of endophytic bacteria as potential biological control agents for oak wilt. Smalla. Appl. 30. M. Fernandez.. Berg. 1999. A. and C. Ecol. 10. Control 29:359–366. 2000. Rev. 16. Dunfield. 29. Physiol. J. C. 1999. Marsh. Morra. 32:241–247.VOL. J. K. Liesack. Characterization of Streptomyces lydicus WYEC108 as a potential biocontrol agent against fungal root and seed rots. 214:215–232. Kinkel. Bacteriol. 2003. J. R. W. J. Increased resistance to Fusarium oxysporum f. Diversity of root-associated bacteria associated with field-grown canola (Brassica napus L. Microbiol. 11. S. 1995. Wilhelm. Christie. L. USA 96:5937–5943. M. and D. Tikhonovich. Siciliano.. 1994. P. 15. 2004. D. 2004 EFFECT OF MICROBES ON WHEAT ROOTS AS DETERMINED BY T-RFLP 6413 4. M. M. J. and D. Appl. Tokala. L. Overbeek. Germida.. 52:487–511. Seib. L. Brooks. 17. 24. Bulk and rhizosphere soil bacterial communities studied by denaturing gradient gel electrophoresis: plant-dependent enrichment and seasonal shifts revealed. Chater. Aust. Environ. M. 16S ribosomal DNA amplification for phylogenetic study. Population dynamics of endophytic bacteria in field-grown sweet corn and cotton. Weisburg. Ecol. G. Arsenault. and C. 12. 63:3233–3241. Coombs. W. Biol. A. Christie. Microbiol. 7. Bailey. Terminal restriction fragment length polymorphism analysis program. FEMS Microbiol. M. D. C. Roskot. Ecol. J. Y. 26:43–50. Hopwood (ed. Warburton. 2001. a web-based research tool for microbial community analysis. Filer. McInroy. Cultivation-independent population analysis of bacterial endophytes in three potato varieties based on eubacterial and Actinomycetes-specific PCR of 16S rRNA genes. J. R. D. de Freitas. and D. Khairallah. and W. H. R. 69:5303–5308. 2000. Siciliano. Microbiol. V. H. B. 1998. Environ. 68:2161–2171. Theoret. Hoisington. J.asm. P.org/ on April 28. Developmental genetics and evolution of symbiotic structures in nitrogen-fixing nodules and arbuscular mycorrhiza. Lukow. Sturz.