Bacillus-based biological control of cotton seedling disease complex

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1 JOURNAL OF PLANT PROTECTION RESEARCH Vol. 54, No. 4 (2014) DOI: /jppr Bcillus-bsed biologicl control of cotton seedling disese complex Mohmmd Ahmed Khiymi 1, Mowd Rgb Omr 2, Kmel Ahmed Abd-Elslm 2 *, Aly Abd El-Hdy Aly 2 1 King Abdulziz City for Science nd Technology (KACST), 6086, Riydh, Sudi Arbi 2 Plnt Pthology Reserch Institute, Agriculturl Reserch Center (ARC), 12619, Giz, Egypt Received: June 27, 2014 Accepted: October 19, 2014 Abstrct: To formulte n efficient nd eco-friendly strtegy for the mngement of cotton seedling disese complex, pot experiments were conducted nd the efficiency of eight Bcillus strins ginst seven fungi involved in the disese were determined. A greenhouse evlution of the interction between fungl isoltes nd Bcillus strins ws crried out. The evlution reveled very highly significnt Bcillus strins x fungl isoltes interction for ll the following prmeters: preemergence dmping-off, postemergence dmping-off, survivl, plnt height, nd dry weight. This interction implies tht single strin of the Bcillus sp. cn be highly effective ginst fungl isolte, but my hve only miniml effects on other fungl isoltes. The results of the present study demonstrted tht Bcillus circulns nd B. cogulns were the most effective strins in controlling cotton seedling disese. Therefore, strins of Bcillus spp. should be tested ginst s mny fungl isoltes s possible. The testing will improve the chnce of identifying Bcillus strins effective ginst severl fungl isoltes. Key words: ntgonstic Bcillus spp., cotton dmping-off complex Introduction Cotton seedling disese complex cuses serious nnul economic losses in mny cotton-producing countries. Rhizoctoni solni, Pythium spp., nd Fusrium spp. re considered the mjor fungi involved in the disese (Wtkins 1981). Sclerotium rolfsii nd M. phseolin my lso be involved in the disese lthough they re less importnt. The use of ntgonistic bcteri showed promising results in controlling soil-borne fungi (Rmrthnm et l. 2011; Smvt et l. 2011; Zim et l. 2013). Pseudomons spp., nd Bcillus spp. re the most common ntgonistic bcteri tht hve been used to control cotton diseses seedling dmping-off (Rmrthnm et l. 2011; El-Hssn et l. 2013; Mnsoori et l. 2013; Sllm et l. 2013; Smvt et l. 2014). Bcillus spp. re erobic or sometimes fculttive bcteri nd ctlse positive. Endospore formtion, universlly found in this group, is thought to be strtegy for survivl in soil, wherein these bcteri predominte nd the endospores mke them resistnt to unfvorble environmentl conditions (Lnd et l. 1997). As ttrctive biocontrol gents nd good plnt-growth-promoting bcteri, Bcillus spp. offer severl dvntges over fluorescent Pseudomons nd other Grm-negtive bcteri. These dvntges include longer shelf life, their bility to form endospores, nd the brod spectrum ctivity of their ntibiotics (Chen et l. 2009; Smvt et l. 2014). Becuse of their disese control properties, Bcillus spp., prticulrly B. subtilis nd its closely relted species, hve been widely studied (Hung et l. 2012). B. subtilis A13 is the best known exmple of biocontrol bcteril strin belonging to the genus Bcillus. This strin ws isolted more thn 25 yers go in Austrli. The strin ws selected bsed on its in vitro inhibitory ctivity ginst nine pthogens nd ws subsequently shown to promote growth of cerels, crrots, nd bedding plnts when it ws pplied s seed tretment (Brodbent et l. 1971; Merrimn et l. 1975). Studies done by Hgedorn et l. (1989) found tht cotton seedlings were initily colonised by bcteri of mny different gener. Popultions quickly reched 10 8 CFU/g of root tissue. As the seson progressed, the bcteri popultions declined while root mss incresed nd the roots becme more woodlike in consistency. There ws no correltion between fungl repressive ctivity of rhizobcteri nd the plnt growth stge from which the isoltes were obtined. B. subtilis GB03, sold tody in the United Sttes s Kodik for control of dmping-off of cotton, is cotton dpted vrint of B. subtilis A13 (Mhffee nd Bockmn 1993). Plebn et l. (1995) reported tht endophytic Bcillius spp. cn survive inside cotton plnts nd re efficient biocontrol gents ginst R. solni nd S. rolfsii under greenhouse conditions. These endophytes reduced the disese index by more thn 50%. Some B. subtilis *Corresponding ddress: kmelbdelslm@gmil.com

2 Bcillus-bsed biologicl control of cotton seedling disese complex 341 strins tht show ntifungl ctivity my ct by inducing tolernce in the host plnt when tested ginst Pythium phnidermtum nd Phytophthor nicotin in soilless tomto nd cucumber culture systems (Grosch et l. 1999). The ctivity of B. cogulns s biologicl control gent ginst fungl plnt pthogens Trichothecium roseum, R. solni, Sclerotini sclerotiorum, Fusrium oxysporum, F. solni, nd F. culmorum ws studied by Czczyk et l. (2002). The effect of 20 isoltes of Bcillus spp., obtined from livestock mnure composts nd cotton-wste composts for in vitro ntgonism ginst F. oxysporum, Phytophtor cpsici, R. solni AG-4, nd S. sclerotiorum ws studied by Kim et l. (2008). Inocultion with both Bcillus pumilus nd Pseudomons lcligenes, cused greter increse in the plnt growth of lentil, the number of pods, nodultion, nd root colonistion by rhizobcteri, nd lso cused reduction of Fusrium wilting to greter degree thn did individul inocultion (Mohd et l. 2010). When using Bcillus spp. nd other bcteri, for their efficcy ginst M. phseolin on soyben plnts, ll tested strins significntly decresed dmping-off, nd decresed rotted nd wilted plnts while incresing helthy plnts (El-Brougy et l. 2009). A wide rnge of plnt pthogens including Erwini crotovor, Fusrium sp., F. oxysporum, Mcrophomin phseolin, Phytophthor, Pythium sp., nd R. solni were controlled by the Bcillus spp. (Jing et l. 2006). The brod-spectrum ntgonistic ctivities of Bcillus re executed by secretion of number of metbolites including ntibiotics, the voltile compound HCN, siderophores, enzymes chitinse nd 1,3-glucnse (Ongen et l. 2007; Chen et l. 2009; Arrebol et l. 2010). The min objective of the present study ws to evlute eight Bcillus strins, isolted from rhizosphere of cotton seedlings, s to their efficiency in controlling soil-borne fungi commonly involved in cotton seedling disese complex under Egyptin conditions. Mterils nd Methods Bcteril strins Soil smples from rhizosphere of cotton (Gossypium brbdense L.) seedlings were collected from different governortes (Ghrbiy, Dkhliy, Giz, Assuit, Beheir, Mini nd Shrkiy). Twenty grms of soil dhering to roots of ech smple were suspended in 80 ml of sterile distilled wter, shken for 30 min on shker, nd then serilly diluted in sterile, distilled wter. One ml from 10 4, 10 5, nd 10 6 dilutions were tken form ech smple nd spred on nutrient glucose gr. The pltes were incubted t 25 C for 3 dys when the developing colonies were isolted in pure cultures. Selected cultures were identified (Tble 1) ccording to Bergey s Mnul of Systemtic Bcteriology (Krieg nd Holt 1984). Fungl isoltes The seven pthogenic isoltes of R. solni, M. phseolin, S. rolfsii, Pythium sp., F. oxysporum, F. solni, nd F. moniliforme used in this study, were originlly isolted from the roots of cotton seedlings infected with dmping-off disese. Isoltion, purifiction, nd identifiction of these fungi were crried out t the Cotton Pthology section of the Plnt Pthology Reserch Institute, the Agriculturl Reserch Centre, Giz, Egypt. Preprtion of fungl inoculum Substrtes for the growth of fungi were prepred in 500-ml glss bottles, ech bottle contined 50 g of sorghum grins nd 50 ml of wter. Contents of bottles were utoclved for 30 min. Fungl inoculum, tken from oneweek-old culture on Potto Dextrose Agr (PDA), ws introduced into bottles nd llowed to colonise sorghum for two weeks. The fungus-sorghum mixture (inoculum) ws stored in plstic bgs t 5 C until use. This study ws crried out by using utoclved cly lom soil. Btches of soil were infested seprtely with inoculum of ech fungus t rtes of 1g/kg soil for R. solni, nd 5 g/kg soil for S. rolfsii, nd Pythium sp. The rtes of F. oxysporum, F. solni, F. moniliforme, nd M. phseolin were 50 g/kg soil. Infested soils were dispensed in 15-cm-dimeter cly pots. The soil ws lso infested with mixture of fungi t the sme previously mentioned rtes. Preprtion of bioformultions Strins were grown in nutrient glucose broth t 30 C for 72 h on shker. The growth ws djusted turbidimetriclly to 10 8 CFU/ml using spectrophotometer 2000 RSP 220 v. 50 Hz. Bcteril cultures were formulted in powder form by mixing 400 ml of cell suspension with 1 kg tlc s crrier, which ws previously utoclved for 30 min Tble 1. Identifiction of bcteril isoltes used in the present study, nd their geogrphic origins Isolte no. Identifiction Geogrphic origin 1 Bcillus cogulns Ghrbiy 2 Bcillus globisporus Dkhliy 3 Bcillus pumilus Giz 4 Bcillus subtilis Assiut 5 Bcillus circulns Beheir 6 Bcillus cereus Giz 7 Bcillus cogulns Mini 8 Bcillus cereus Shrkiy

3 342 Journl of Plnt Protection Reserch 54 (4), 2014 for 2 successive dys. Ten grms of crboxy methyl cellulose (CMC) were dded to 1 kg of the crrier nd mixed thoroughly. The ph of ll mterils ws djusted to 7.0 by dding clcium crbonte. The bcteril popultion ws ssessed s CFU/g tlc (Gordon-lennox et l. 1987). Greenhouse pot experiment Slightly moist seeds of cultivr Giz 89 were treted with the powdered inoculum of ech Bcillus strin t rte of 10 g/kg seeds. The treted seeds were then thoroughly shken in plstic bgs before being plnted in soil previously infested with the tested fungi. The seeds were plnted t rte of 10 seeds/pot. The pots were rndomly distributed on greenhouse bench. The temperture regime rnged from 19±4 to 30±4 C. The experiment inculded three control tretments: Monceren-treted seeds in infested soil, untreted seeds in infested soil, nd untreted seeds in utoclved soil. The percentges of preemergence nd postemergence dmping-off were recorded 15 dys from sowing while the percentge of surviving seedling, dry weight (mg/plnt), nd plnt height (cm/ /plnt) were recorded 45 dys from sowing. All tests were repeted once with lmost the sme results. Sttisticl nlysis of the dt The experimentl design in the present study ws Rndomized Complete Block with five replictions. Anlysis of vrince (ANOVA) of the dt ws performed with the MSTAT-C Sttisticl Pckge. Lest Significnt Difference (LSD) ws used to compre tretment mens within ech fungus (α = 0.05 or α = 0.01). Percentge dt were trnsformed into rc sine ngles or x before crrying out ANOVA to produce the pproximte constnt vrince. Results Fungi, tretments, nd fungi tretment interctions were significnt sources of vrition in most of the tested prmeters (Tble 2). Reltive contributions of fungi, tretments, nd their interctions to vrition in the tested prmeters re shown in figure 1. Due to the significnt tretment fungus interction (Tble 2), LSD ws used to compre between tretments within ech fungus (Tble 3). These comprisons showed tht ll the tested fungi nd their mixtures were pthogenic in the preemergence stge tretments 1, 3, 5, nd 8 were highly effective in controlling preemergence dmping-off cused by R. solni s they decresed infection by 88.9, 83.3, 94.4, nd 94.4%, respectively. Strins 5 nd 8 were more efficient thn the fungicide Monceren in controlling R. solni t the preemergence stge. Strin 4 completely suppressed M. phsolin, while strin 8 showed complete filure in controlling M. phsolin. The other strins showed vrible effects. Strins 4 nd 7 were effective in controlling S. rolfsii while strin 6 ws completely ineffective. Strin 6 ws effective ginst F. oxysporum, but strin 6 incresed infection by F. solni. Strin 2 incresed infection by F. moniliforme, while strins 7 nd 2 decresed infection by the mixture. Strin 2 ws the most effective strin ginst the mixture followed by strin no. 7 (efficiency ws 82.35% nd 76.47%, respectively). Strins 3 nd 7 were effective in controlling Pythium sp., while strins 1, 6, nd 8 were ineffective. Strins 3 nd 7 were s effective s the fungicide. Dt in tble 4 indicted tht ll the tested fungi nd their mixtures were pthogenic in the postemmergence stge. Tretments 1, 6, 7 nd 8 were more efficient thn the fungicide (tretment 9) in controlling M. phsolin in the postemergence stge s they decresed postemergence by 77.7, 88.9, 55.9, nd 55.9%, respectively, while the efficiency of the fungicide (no. 9) ws 40.0%. All the eight strins were highly effective in controlling postemergence dmping-off cused by S. rolfsii nd the efficiency of the tretments ws higher thn the efficiency of the fungicide. Strin no. 3 completely suppressed S. rolfsii. Dt shown in tble 4 indicte tht ll tretments were effective in decresing the postemergence dmping-off cused by Pythium sp. nd F. oxysporum. Strin no. 1 ws s effective s the fungicide in controlling Pythium sp. At the sme time, strin no. 5 ws more efficient thn the fungicide in controlling both fungi. Strins 5, 6, 7, nd 8 were highly effective in controlling postemergence dmping-off cused by F. solni since Fig. 1. Reltive contribution of tretments, fungi, nd their interction to vrition in cotton seedling disese vribles (cv. Giz 89) under greenhouse conditions. The reltive contribution ws clculted s the percentge of the sum squres of the explined (model) vrition

4 Bcillus-bsed biologicl control of cotton seedling disese complex 343 Tble 2. Anlysis of vrince of the effect of tretments, fungi, nd their interction on cotton seedling disese vribles (cv. Giz 89) under greenhouse conditions Source df MS F vlue P vlue Preemergence dmping-off Repliction Fungi (F) Tretment (T) F T Error Postemergence dmping-off Repliction Fungi (F) Tretment (T) 10 3, F T Error Survivl Repliction Fungi (F) Tretment (T) 10 4, F T Error Plnt height Repliction Fungi (F) Tretment (T) F T Error Dry weight Repliction Fungi (F) Tretment (T) 10 19, F T Error repliction is rndom, while ech of the tretments nd fungi re fixed df degrees of freedom; MS men squre these strins decresed infection by 92.6, 77.8, 96.3, nd 88.9 while efficiency of the fungicide ws On the other hnd, strins 1, 2, 3, 4, nd 8 were highly effective in controlling postemergence dmping-off cused by F. moniliforme, nd these strins were more effective thn the fungicide. Strins 2 nd 7 were effective ginst the mixture s they decresed infection by 79.51% nd 79.51%. Strins 3 nd 4 followed in effectiveness. At the sme time, strins 2 nd 7 were s effective s the fungicide (no. 9) in controlling postemergence dmping-off cused by the mixture. Dt shown in tble 5 indictes tht ll bcteril strins incresed the survivl number when compred with tretment no. 10 (infested soil). Strins 1, 3, 5, nd 8 were highly effective in incresing survivl in the cse of R. solni where the survivl numbers were 67.5, 67.5, 80.0, nd 85.0, respectively. Strin no. 8 ws equivlent (s high s 85.0) to the fungicide in its effect on the percentge of survivl. On the contrry, the bcteril strins 1, 4, 6, nd 7 were more effective in incresing survivl thn the fungicide when used for controlling M. phseolin. Thus, the percentges of survivl of strins 1, 4, 6, nd 7 were 75.0, 77.5, 850, nd 75.0, respectively while tht of tretment no. 9 (the fungicide) ws All the eight strins were more effective thn the fungicide in incresing the survivl when used to control S. rolfsii, F. oxysporum, nd F. moniliforme. Regrding F. solni, tretments 1, 2, 3, 5, 6, 7, nd 8 were less effective thn the fungicide in incresing survivl. On the other hnd, strin no. 4 ws more effective thn Monceren in incresing survivl (to 80.0) while Monceren incresed it to As to the fungl mixture, tretments 2 nd 7 were highly effective in incresing survivl. Their percentges were 87.5 nd 85.0 while tht of the fungicide ws Dt in tbles 6 nd 7 show tht ll strins cused highly significnt increses in plnt height nd dry weight with ll the fungi nd the mixture, when compred with the control (infested soil). For exmple, when strin no. 1 ws used for controlling R. solni, plnt height incresed to cm, wheres plnt height incresed to cm when infested soil (no. 10) ws used. Strin no. 1 incresed dry weight to mg while with the use of infested soil, the dry weight ws 69.8 mg. Strin no. 4 ws effective in improving plnt height nd dry weight when used for controlling M. phsolin nd S. rolfsii. With the use of strin 4, plnt height ws cm nd cm, respectively, while dry weight ws mg nd mg. On the other hnd, strin no. 4 ws effective in incresing plnt

5 344 Journl of Plnt Protection Reserch 54 (4), 2014 Tble 3. Effect of tretments, fungi, nd their interction on preemergence dmping-off of cotton seedlings (cv. Giz 89) under greenhouse conditions Tretment Fungi R. solni M. phseolin S. rolfsii Pythium sp. F. oxysporum F. solni F. moniliforme mixed % b trnsformed % trnsformed % trnsformed % trnsformed % trnsformed % trnsformed % trnsformed % trnsformed tretments from 1 to 8 re bcteril strins shown in tble 1, tretment 9 is Monceren, tretment 10 is infested soil, nd tretment 11 is utoclved soil b percentge dt were trnsformed into x before crrying out ANOVA to pproximtely produce constnt vrince LSD (trnsformed dt ) for tretment fungi interction = (α = 0.05) or 1.81 (α = 0.01) Tble 4. Effect of tretments, fungi, nd their interction on postemergence dmping-off of cotton seedlings (cv. Giz 89) under greenhouse conditions Tretment Fungi R. solni M. phseolin S. rolfsii Pythium sp. F. oxyspoum F. solni F. moniliforme mixed % b trnsformed % trnsformed % trnsformed % trnsformed % trnsformed % trnsformed % trnsformed % trnsformed tretments from 1 to 8 shown in tble 1, tretment 9 is Monceren, tretment 10 is infested soil, nd tretment 11 is utoclved soil b percentge dt were trnsformed into rc sine ngles before crrying out ANOVA to pproximtely produce constnt vrince LSD (trnsformed dt) for tretment fungi interction = (α = 0.05) or (α = 0.01)

6 Bcillus-bsed biologicl control of cotton seedling disese complex 345 Tble 5. Effect of tretments, fungi, nd their interction on survivl of cotton seedlings (cv. Giz 89) under greenhouse conditions Fungi Tretment R. solni M. phseolin S. rolfsii Pythium sp. F. oxysporum F. solni F. moniliforme mixed % b trnsformed % trnsformed % trnsformed % trnsformed % trnsformed % trnsformed % trnsformed % trnsformed tretments from 1 to 8 re the bcteril strins shown in tble 1, tretment 9 is Monceren, tretment 10 is infested soil, nd tretment 11 is utoclved soil b percentge dt were trnsformed to into rc sine ngles before crrying out ANOVA to pproximtely produce constnt vrince LSD for tretment fungi interction = 8.60 (α = 0.05) or (α = 0.01) Tble 6. Effect of tretments, fungi, nd their interction on plnt height (cm/plnt) of cotton seedlings (cv. Giz 89) under greenhouse conditions Fungi Tretment R. solni M. phseolin S. rolfsii Pythium sp. F. oxysporum F. solni F. moniliforme mixed tretments from 1 to 8 re the bcteril strins shown in tble 1, tretment 9 is Monceren (fungicides), tretment 10 is infested soil, nd tretment 11 is utoclved soil LSD for tretment fungi interction = 4.14 (α = 0.05) or 5.46 (α = 0.01)

7 346 Journl of Plnt Protection Reserch 54 (4), 2014 Tble 7. Effect of tretments, fungi, nd their interction on dry weight (mg/plnt) of cotton seedlings (cv. Giz 89) under greenhouse conditions Fungi Tretment R. solni M. phseolin S. rolfsii Pythium sp. F. oxysporum F. solni F. moniliforme mixed tretments from 1 to 8 re the bcteril strins shown in tble 1, tretment 9 is Monceren, tretment 10 is infested soil, nd tretment 11 is utoclved soil LSD for tretment fungi interction = (α = 0.05) or Ongen (α = 0.01) height when used to control Pythium sp. while it ws not effective in improving dry weight. Strins 2 nd 6 were effective in incresing dry weight nd plnt height in the cse of Pythium sp. The results of the mixture showed tht strins 3, 5, 7, nd 8 were effective in incresing plnt height to 30.75, 28.93, 28.90, nd cm, respectively, while the infested soil (no. 10) ws 9.35 cm. Strins 1, 3, nd 6 were lso useful in incresing dry weight. In generl, ll tretments were more effective thn the fungicide in incresing both plnt height nd dry weight. Discussion The genus Bcillus is one of the the most importnt biocontrol gents used ginst insect plnt pthogens. Bcillus hs been shown to be effective nd ws commercilly pplied ginst plnt pthogenic fungi (Jing et l. 2006; Jin 2008). For instnce, B. pumilus decresed disese incidence cused by M. phseolin in chickpe plnts (Akhtr nd Siddiqui 2008). In the current investigtion, eight different strins of Bcillus spp. were evluted s biocontrol ginst seven fungi involved in seedling disese complex of cotton nd their mixture. These results re in greement with the erly reports tht this group of bcteri re responsible for the biocontrol ctivity in rhizosphere of cotton seedlings (Aly et l. 1996; Wn et l. 2012). The results showed tht strins 1, 3, 5, nd 8 were effective in controlling R. solni in the preemergence stge, while strins 4, 5, nd 8 were effective in the postemergence stge. These finding indicted tht strins 5 nd 8 were ble to mintin their efficiency in the preemergence nd postemergence stges. This phenomen ws lso observed by Ask nd Shod (1996) nd could be due to the presence of certin fungisttic metbolites secreted by these bcteril strins in preemergence nd postemergence stges. For exmple, B. subtilis strin NCD-2 produced fengycin lipopeptide, nd the fengycin plyed primry role in inhibiting the growth of R. solni in vitro, in ddition to suppressing cotton seedling dmping-off disese in vivo (Guo et l. 2013). Results lso showed tht the bcteril strins my chnge their efficiency when used for controlling different fungi. For exmple, strin 8 showed complete filure in controlling M. phsolin nd S. rolfsii while the sme strin ws effective in controlling R. solni. This result implies tht the bcteril strin my succeed in controlling some fungi, while it my fil to suppress other fungi (Hrris et l. 1994). The high significnce (p = ) of the interction of Bcillus isoltes M. phseolin isoltes suggests tht single isolte of the ntgonist cn be highly effective ginst n isolte of M. phseolin, but my hve miniml effects on the other isoltes of M. phseolin (Omr et l. 2013). There were significnt increses in survivl nd reduced preemergence dmping-off showed by B. cogulns nd B. cereus (Aml-Asrn 2001). The results of the present study showed tht some bcteri strins my induced or increse the emergence of dmping-off in cotton seedling. For exmple, strin 2 nd strin 3 increse the premergence dmping-off disese ginst F. moniliforme nd F. solni, respectively. The fomentioned results indicted tht these bcteril strins my be deleterious to plnts, by producing toxic metbolites without prsitising plnt tissues (Schippers et l. 1987). The results of our studies lso showed tht Bcillus strins sometimes promote plnt height nd dry weight of cotton seedlings. These effects my be due to the fct tht Bcillus spp. such s B. subtilis is known to produce growth-promoting fctors (Swin nd Ry 2009; López- -Vldez et l. 2011). Bcillus strins protect the roots from pthogen invsion nd promote plnt growth by solubilising soil phosphorus nd producing hydrolytic enzymes, ntibiotics, nd plnt growth hormones (Rz et l. 2009; Ling et l. 2010; Abdel-Ftth et l. 2011).

8 Bcillus-bsed biologicl control of cotton seedling disese complex 347 The introduction of P. uerofciens s biocontrol gent ginst R. solni cn result in n incresed cotton yield, decresed chemicl fungicide ppliction, nd protection of the griculturl environment nd biologicl resources (Smvt et l. 2014). The results of the present study demonstrted tht B. circulns nd B. cogulns were the most effective strins in controlling cotton seedling disese. Acknowledgements This work ws prtilly supported by the Agriculturl Reserch Centre, Plnt Pthology Reserch Institute, Giz, Egypt. References Abdel-Ftth G.M., El-Hddd S.A., Hfez E.E., Rshd Y.M Induction of defense responses in common ben plnts by rbusculr mycorrhizl fungi. Microbiol. Res. 166 (4): Akhtr S.M., Siddiqui Z.A Glomus intrrdices, Pseudomons lcligenes nd Bcillus pumilus, effective gents for the control of root-rot disese complex of chickpe Cicer rietinum L. J. Gen. 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