شناسایی مورفولوژیکی و مولکولی فوزاریوم‌های (Fusarium spp.) اِندوفیت ریشه جودره (Hordeum spontaneum) و شلمی (Rapistrum rugosum)

نوع مقاله : مقالات پژوهشی

نویسندگان

1 گروه زراعت و اصلاح و نباتات، پردیس کشاورزی و منابع طبیعی دانشگاه تهران، کرج

2 گروه گیاه‌پزشکی، پردیس کشاورزی و منابع طبیعی دانشگاه تهران، کرج

3 پژوهشگاه بیوتکنولوژی

چکیده

اندوفیت­ها میکروارگانیسم­های درون بافت­های گیاهی هستند و اغلب بدون اینکه علایم بیماری قابل مشاهده­ای را بروز دهند، برهمکنش­های مختلفی با میزبان خود دارند. در این تحقیق، فوزاریوم­های اندوفیت ریشه جودره (Hordeum spontaneum) و شلمی (Rapistrum rugosum) ارزیابی شدند. برای این منظور بذر علف­های هرز بیان شده روی خاک مزارع گندم نمونه‌برداری شده از استان­های البرز، تهران، خراسان رضوی، خراسان شمالی، خوزستان، فارس، قزوین،کرمانشاه و گلستان در شرایط گلخانه درون گلدان کشت شدند. گیاهان در مرحله 6-5 برگی برداشت شده و ریشه­ها پس از قطع شدن از محل طوقه، در تکه­های کوچک روی محیط کشت اختصاصی فوزاریوم، پنتاکلرو نیترو بنزن پپتون آگار کشت داده شد. جدایه­ها پس از خالص­سازی براساس ویژگی­های ریخت­شناسی و توالی ژن TEF 1-αشناسایی شدند. قارچ Fusarium equiseti گونه غالب در اکثر مناطق بود که از هر دو گونه علف­هرز جداسازی شد. به علاوه قارچ­های F. redolens و F. acutatum فقط از جودره وF. oxysporum  و F. torulosum فقط از شلمی جداسازی شدند. آگاهی از طیف میکروبی اندوفیت علف­های هرز، می­تواند در درک بهتر برهمکنش­های میان علف هرز-میکروارگانیسم­ها کمک کرده و حتی ممکن است فرصت­هایی در جهت بهره­برداری از این عوامل در برنامه­های کنترل زیستی فراهم نماید.

کلیدواژه‌ها

موضوعات


عنوان مقاله [English]

Morphological and Molecular Identification of Fusarium spp. as Root Endophytes of Spontaneous Barley (Hordeum spontaneum) and Turnipweed (Rapistrum rugosum)

نویسندگان [English]

  • Farshid Nooralvandi 1
  • Hasan Alizadeh 1
  • Hosein Saremi 2
  • Gholamreza Salehi Jozani 3
1 College of Agriculture and Natural Resources of University of Tehran, Karaj
2 Department of Plant Protection, College of Agriculture and Natural Resources of University of Tehran, Karaj
3 Agriculture Biotechnology Research Center
چکیده [English]

Introduction: Endophytic microorganisms are present inside plant tissues without evident disease symptoms, but they have different interactions with their host. These microorganisms have revealed a great potential to be exploited as a biological control agent of troublesome weeds. Endophytic microorganisms in association with weed roots can act as a soil application herbicide through toxin secretion. These agents in most cases do not kill target weed but instead, have an ability to suppress them noticeably, so the crop can compete with companion weeds very successfully. Unfortunately, the endophytic microorganisms of weeds are less studied and such studies is very limited. So, this experiment was conducted to study root endophytic Fusarium species of Hordeum spontaneum and Rapistrum rugosum as serious weeds of winter cereals of our country. Fusarium species are one of the most important host-specific microorganisms in the biological control program of weeds.
Material and Methods: The weeds planted in pots filled with the soil of wheat farms sampled from Alborz, Tehran, Khorasan shomali, Khuzestan, Fars, Qazvin, Kermanshah and Golestan provinces (Iran), under greenhouse condition. The plant harvested at 5–6 leaf stage, roots were cut from the crown and sliced into small pieces. Then the pieces of root were cultured on specific Fusarium medium, Penta chloro Nitro Benzene Peptone Agar (PPA). Fusarium spp. grown on the medium, sub-cultured onto the Water Agar (WA) medium. After 24-72 h hyphae tip of the isolates translocated onto the Potato Dextrose Agar (PDA) medium and let them to fully grow and pure isolates obtained.  Some morphological details of the species e.g. chlamydospore formation and physical characteristics of it, color and diameter of the colony (after 72 h) were assessed according to their growth habit on PDA medium. Each species translocated onto Carnation Leaf Agar (CLA) and Synthetic Nutrient Poor Agar (SNA) mediums to evaluate other necessary morphological characteristics. Sporodochium formation and physical characteristics of macroconidia on CLA, and microconidia formation and physical characteristics of microconidia on SNA, also assessed. Isolates were identified based on molecular data that were generated for TEF 1-α gene, following PCR amplification as well.  Pro Chromas (1.7.6 version) and Editseq (5.01 version) softwares were used to edit the sequences before further processing. Edited sequences were blasted in NCBI (http://www.ncbi.nlm.nih.gov/blast/) and compared to those sequences that already existed in the database to find similarities and molecularly identification of species. Selected sequences of the study and those isolates from the database were alignment using Clustal X (version 2.1) software and finally phylogenic tree was drawn using MEGA6 (molecular evolutionary genetics analysis). And appropriate outgroup was exploited to sequences analysis. Finally, maximum composite likelihood method was used to analyze and assessment of nucleotide sequence of gene regions of different isolates. With taking into account of all these information together, we were able to identify all isolated Fusarium species with confidence.
Results and Discussion: Fusarium equiseti was the dominant species recovered in several regions, while the other Fusarium species were site and species-specific. F. equiseti was recovered from the root of H. spontaneum and R. rugosum plants cultured in the soil of Fars and Golestan. This species also recovered from the root of H. spontaneum and R. rugosum cultured in the soil of Khouzestan and Qazvin provinces respectively. In addition, F. redolens and F. acutatum were recovered from H. spontaneum root cultured in the soil of Khorasan Shomali and Qazvin provinces respectively. On the other side, F. torulosum and F. oxysporum were recovered from R. rugosum root cultured in the soil of Kermanshah and Tehran respectively. Totally 10 isolates of five different species of Fusarium recovered from the root of H. spontaneum and R. rugosum. It shows probably a lot of Fusarium species (and maybe other microorganisms) are in contact with weeds root which can be utilized as their potential biological agent, but the potential is not studied to a great extent. There are many reports that show many species of Fusarium are exploited as biological agents of different weed species. This is because of the host specificity of the fungus Fusarium which turns it to a suitable and efficient agent in the biological control program of serious hard to chemical control weeds.
Conclusion: Many grass weeds are very hard to manage with conventional methods especially chemical means. We have to look for other alternative solutions to overcome them. Biological control of weeds is a promising approach to wisely control of weeds, especially those that are very resistance prone or hardly controlled with available herbicides. Exploiting this approach needs proper and diverse control means. Microorganisms are a rich source of biological agents of weeds that less attention has been paid to them. Endophytic microorganisms of weeds are a new generation of biological agents that studies have been focused on them recently. The first step to choose an efficient agent is having a deep knowledge about what kind of species are living with them. In fact, detailed knowledge about endophytic microorganism of weeds can aid in better understanding of the nature of interactions between weed-microbe and how to exploit these as agents in weed biological control programs.
 

کلیدواژه‌ها [English]

  • Fusarium equiseti
  • fungus
  • TEF
  • Weed
  • wheat
1-       Ahluwalia A.D. 2007. Bioherbicides: an eco-friendly approach to weed management. Current Science 92: 10-11.
2-       Altschul S.F., Madden T.L., Schäffer A.A., Zhang J., Zhang Z., Miller W., and Lipman D.J. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25: 3389-3402.
3-       Andrews M., Cripps, M.G., and Edwards G.R. 2012. The potential of beneficial microorganisms in agricultural systems. Annals of Applied Biology 160: 1-5.
4-       Beest D.O., Yang X.B., and Cisar C.R. 1992. The status of biological control of weeds with fungal pathogens. Annual Review of Phytopathology 30: 637-657.
5-       Boari A., and Vurro M. 2004. Evaluation of Fusarium spp. and other fungi as biological control agents of broomrape (Orobanche ramosa). Biological Control 30: 212-219.
6-       Caesar A.J. 1999. Insect/pathogen interactions are the foundation of weed biocontrol. In: Program Abstracts,XInternational Symposium on Biological Control of Weeds, USDAARS and Montana State University, Bozeman, MT. Pp. 53.
7-       Chandler D., Bailey A.S., Tatchell G.M., Davidson G., Greaves J., and Grant W.P. 2011. The development, regulation and use of bio-pesticides for integrated pest management. Philosophical Transactions of the Royal Society B: Biological Sciences 366: 1987-1998.
8-       Ciotola M., Watson A.K., and Hallett S.G. 1995. Discovery of an isolate of Fusarium oxysporum with potential to control Striga hermonthica in Africa. Weed Research 35: 303-309.
9-       Davari M., Babai-Ahari A., Arzanlou M., Zare R., D Van Diepeningen A., and De Hoog G.S. 2014. Morphological and molecular characterization of three new Fusarium species associated with inflorescence of wild grasses for Iran. Rostaniha 14: 124-134. (In Persian with English abstract)
10-   Fröhlich J., Morin L., Gianotti A., and Webster R. 1999. Exploring the host range of Fusarium tumidum, a candidate bioherbicide for gorse and broom in New Zealand. In: Program Abstracts, Xth International Symposium on Biological Control of Weeds. Pp. 121.
11-   Garibaldi A., Gilardi G., and Gullino M.L. 2006. Evidence for an expanded host range of Fusarium oxysporum f. sp. raphani. Phytoparasitica 34: 115–121.
12-   Geiser D.M., del Mar Jiménez-Gasco M., Kang S., Makalowska I., Veeraraghavan N., Ward T.J., and O'donnell K. 2004. FUSARIUM-ID v.1.0: A DNA sequence database for identifying Fusarium. European Journal of Plant Pathology 110: 473-479.
13-   Hatami Moghadam Z., Gherkhlou J., De Prado R., and Sadeghi Pour H.R. 2016. Tracing wild mustard (Sinapis arvensis) and Turnipweed (Rapistrum rugosum) biotypes resistance to Tribenuron methyl herbicide in wheat fields of Golestan province and introduction a new method for determination resistant populations of these weeds. Iranian Journal of Crop Protection 30: 347-358. (In Persian with English abstract)
14-   Heap I. (2014). Global perspective of herbicide‐resistant weeds. Pest Management Science 70(9): 1306-1315.
15-   Hirsch P.R., and Mauchline T.H. 2012. Mutualism: plant-microorganism interactions. Pp. 43-55 In: Ogilvie, L.A., Hirsch, P.R. (eds). Microbial Ecological Theory. Norfolk, United Kingdom: Caister Academic Press.
16-   Iasur Kruh L., Lahav T., Abu-Nassar J., Achdari G., Salami R., Freilich S., and Aly R. 2017. Host-parasite-bacteria triangle: the microbiome of the parasitic weed Phelipanche aegyptiaca and tomato-Solanum lycopersicum (Mill.) as a host. Frontiers in Plant Science 8: 269.
17-   Jia M., Ming Q.L., Zhang Q.Y., Chen Y., Cheng N., Wu W.W., and Qin L.P. 2014. Gibberella moniliformis AH13 with antitumor activity, an endophytic fungus strain producing triolein isolated from adlay (Coix lacryma-jobi: Poaceae). Current Microbiology 69: 381-387.
18-   Klironomos J.N. 2003. Variation in plant response to native and exotic arbuscular mycorrhizal fungi. Ecology 84: 2292-2301.
19-   Kremer R.J. 2013. Interactions between the plants and microorganisms. Allelopathy Journal 31: 51-70.
20-   Kremer R.J., and Kennedy, A.C. 1996. Rhizobacteria as biocontrol agents of weeds. Weed Technology, 10: 601-609.
21-   Kroschel J., Mueller-Stoever D., Elzein A., and Sauerborn J. 2000. The development of mycoherbicides for the management of parasitic weeds of the genus Striga and Orobanche-a review and recent results. In: Proceedings of the X International Symposium on Biological Control of Weeds. Pp. 139. Bozeman, MT: Montana State University.
22-   Ladygina N., and Hedlund K. 2010. Plant species influence microbial diversity and carbon allocation in the rhizosphere. Soil Biology and Biochemistry 42: 162-168.
23-   Lakshmi V., Kumari S., Singh A., and Prabha C. 2015. Isolation and characterization of deleterious Pseudomonas aeruginosa KC1 from rhizospheric soils and its interaction with weed seedlings. Journal of King Saud University-Science 27(2): 113-119.
24-   Leslie J.F., Summerell B.A., and Bullock S. 2006. The Fusarium laboratory manual (Vol. 2, No. 10). Ames, IA.
25-   Lu P., Gilardi G., Gullino M.L., and Garibaldi A. 2010. Biofumigation with Brassica plants and its effect on the inoculum potential of Fusarium yellows of Brassica crops. European Journal of Plant Pathology 126: 387-402.
26-   Maciá-Vicente J.G., Jansson H.B., Abdullah S.K., Descals E., Salinas J., and Lopez-Llorca L.V. 2008a. Fungal root endophytes from natural vegetation in Mediterranean environments with special reference to Fusarium spp. FEMS Microbiology Ecology 64: 90-105.
27-   Maciá-Vicente J.G., Jansson H.B., Mendgen K., and Lopez-Llorca L.V. 2008b. Colonization of barley roots by endophytic fungi and their reduction of take-all caused by Gaeumannomyces graminis var. tritici. Canadian Journal of Microbiology 54: 600-609.
28-   Márquez S.S., Bills G.F., and Zabalgogeazcoa I. 2007. The endophytic mycobiota of the grass Dactylis glomerata. Fungal Divers 27: 171-195.
29-   Mendes R., Garbeva P., and Raaijmakers J.M. 2013. The rhizosphere microbiome: significance of plant beneficial, plant pathogenic, and human pathogenic microorganisms. FEMS Microbiology Reviews 37: 634-663.
30-   Montazeri M., Zand E., and Baghestani M.A. 2003. Weeds and their control in wheat fields of Iran. Weed Research Department of Research Institute of Pest and Diseases. 85 pp.
31-   Motlagh M.R.S. 2011. Fusarium equiseti (Corda) Saccardo as biological control agent of barnyardgrass (Echinochloa crus-galli L.) in rice fields. Journal of Food, Agriculture & Environment 9: 310-313.
32-   Mrema E., Shimelis H., and Laing M. 2019. Combining ability of yield and yield components among Fusarium oxysporum f. sp. strigae-compatible and Striga-resistant sorghum genotypes. Acta Agriculturae Scandinavica, Section B—Soil & Plant Science 1-14.
33-   Nachtigal G.D.F., and Pitelli R.A. 1999. Fusarium sp. as a potential biocontrol agent for Egeria densa and E. najas. In: Program Abstracts, X International Symposium on Biological Control of Weeds. Pp. 68.
34-   National Center for Biotechnology Information (NCBI)[Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] - [cited 2017 Apr 06]. Available from: https://www.ncbi.nlm.nih.gov/.
35-   Nazer Kakhaki S.H., Montazeri M., and Naseri B. 2017. Biocontrol of broomrape using Fusarium oxysporum f. sp. orthoceras in tomato crops under field conditions. Biocontrol Science and Technology, 27: 1435-1444.
36-   Park J.M., Radhakrishnan R., Kang S.M., and Lee I.J. 2015. IAA producing Enterobacter sp. I-3 as a potent bio-herbicide candidate for weed control: a special reference with lettuce growth inhibition. Indian Journal of Microbiology 55: 207-212.
37-   Payedar S., Zand E., Baghestani M.A., and Jamali M.R. 2009. Investigation efficacy of native and foreign Fenoxaprop-p-ethyl for controlling wild Oat (Avena ludoviciana). Iranian Journal of Weed Research 1: 45-55. (In Persian with English abstract)
38-   Shirdashtzadeh M. 2014. Deleterious rhizobacteria as weed biological control agent: Development and constraints. Asian Journal of Microbiology, Biotechnology and Environmental Sciences 16: 561-574.
39-   Sindhu S.S., Khandelwal A., Phour M., and Sehrawat A. 2018. Bioherbicidal potential of rhizosphere microorganisms for ecofriendly weed management. In Role of Rhizospheric Microbes in Soil (pp. 331-376). Springer, Singapore.
40-   Tamura K., Nei M., and Kumar S. 2004. Prospects for inferring very large phylogenies by using the neighbor-joining method. Proceedings of the National Academy of Sciences (USA) 101:11030-11035.
41-   Tamura K., Stecher G., Peterson D., Filipski A., and Kumar. S. 2013. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular Biology and Evolution 30: 2725-2729.
42-   Thompson J.D., Gibson T.J., Plewniak F., Jeanmougin F., and Higgins D.G. 1997. The Clustal X Windows interface: flexible strategies for multiply sequence alignment aided by quality analysis tools. Nucleic Acids Research 24: 4876–4882.
43-   Tranel P.J., Gealy D.R., and Kennedy A.C. 1993. Inhibition of downy brome (Bromus tectorum) root growth by a phytotoxin from Pseudomonas fluorescens strain D7. Weed Technology 7: 134-139.
44-   Van Lenteren J.C., Bolckmans K., Köhl J., Ravensberg W.J., and Urbaneja A. 2018. Biological control using invertebrates and microorganisms: plenty of new opportunities. BioControl 63: 39-59.
45-   Wardle D.A., Yeates G.W., Williamson W., and Bonner K.I. 2003. The response of a three trophic level soil food web to the identity and diversity of plant species and functional groups. Oikos 102: 45-56.
46-   Zand E., Baghestani M.A., Nezam Abadi N., Minbashi Moeini M., and Hadizadeh M.H. 2009. A review on the last list of herbicides and the most important weeds of Iran. Iranian Journal of Weed Research 2: 83-100. (In Persian with English abstract)
47-   Zhong S., and Steffenson B.J. 2001. Virulence and molecular diversity in Cochliobolus sativus. Phytopathology 91: 469-476.