31
Effect of Some Biocontrol Agents Against Rhizoctonia solani in vitro
Khalid A. Asiry and Ibrahim Mohamed Ali
Department of Arid Land Agriculture, King Abdulaziz University, Jeddah, Saudi Arabia [email protected]
Abstract.Root rot of alfalfa caused by Rhizoctonia solani is considered one of the most important diseases, which annually occurred in many plants and causes great losses in yield production. So the present study aimed to find the best strategy to control or at least reduce disease severity. The Results showed that the fungal isolates were identified by using the morphological features of mycelia as R.
solani. Effect of certain antagonistic bio-agents fungi to inhibit the growth of R. solani has been studied under laboratory. In general isolate No. 2002, followed by 2008 and 2004, which recorded (60.5, 47.2and 33.1%), respectively In this respect, the highest inhibition of R. solani growth was occurred by isolate No.5001, followed by 5007 and 5009, 5002, which recorded (54.56, 449.39, 46.26 and 46.26 %), respectively. Eight bacterial strains were tested against growth of Rhizoctonia sp was investigated in vitro. demonstrated that all tested bacteria significantly inhibited growth of Rhizoctonia sp in vitro the highest inhibition of R. solani growth was occurred by isolate No.1001, followed by 1016 and 1010, which recorded (59.25, 52.76 and 47.16 %), respectively. While, the least inhibition of Rhizoctonia growth was occurred by isolate No.1009, which recorded (31.47%).
Ten yeast strains were tested against growth of the pathogen were investigated in vitro demonstrated that all tested yeast significantly inhibited growth of the pathogen in vitro. In conclusion, we could say that Bio- control agents could be used as an eco-friendly approach to effectively control the disease and may be advised to the farmers for profitable organic farming.
Keywords: Rhizoctonia, Biological control, Alfaalf.
1. Introduction
Biological control can be defined as the use of an organism to reduce the population density of another organism and thus includes the control of animals, weeds and diseases. In this article, we focus on the biological control of arthropods, which De Bach (1964) defined as
‘the study and uses of parasites, predators and pathogens for the regulation of host (pest) densities’. This definition establishes two of the main principles of biological control. Firstly, in nature, most organisms are consumed by other organisms, which in many cases leads to drastic reductions in the population of the prey species;
in biological control, man exploits this ‘natural control’ to suppress the numbers of pest
species. Secondly, biological control reduces rather than eradicates pests, such that the pest and natural enemy remain in the agro- ecosystem at low densities. A number of important pests can be kept at a low population density by biological control over long periods of time. In other cases, populations of pests are significantly reduced by natural enemies, but repeated releases or additional methods are needed to achieve an adequate level of control.
These methods include, for example, resistant plants, cultural techniques, physical barriers, semiochemicals and, as a last resort, the use of selective chemicals; this is the fundamental philosophy of integrated pest management (Stern et al. 1959).
Many biological control schemes use predatory insects and mites, insects that parasitize other insects (parasitoids) or nematodes, targeted against insect and mite pests; these are the so-called ‘macrobial’
agents. There are also various ‘microbial’
agents (bacteria, viruses and fungi) that have been developed and applied in arthropod biological control. Herbivorous insects and mites have also been used in the biological control of weeds (Bellows & Fisher 1999; van Lenteren 2003). The present work aimed to study the following points -Isolation and identification of pathogenic fungi causing root rot disease of alfa alfa plants. -Isolation and identification of certain bio-agents - Effect of treatment with certain bio- control agents on growth of the pathogen under laboratory conditions.
2. Materials and Methods
2.1 Isolation and Identification of Alfalfa Root Rot
Samples of diseased alfalfa plants showing typical symptoms of root rot diseases of soybean were collected from Hada Elsham.
To isolate the causal pathogen, crown, roots and stem lesions of the affected tissues were cut into small pieces (1-cm-long) and washed separately under running tap water, surface sterilized by immersing them for 1-2 minutes in 2.0% sodium hypochloride solution, rinsed three times in sterilized distilled water and dried with sterilize filter papers. The surface sterilized plant pieces were placed on Potato Dextrose Agar medium (PDA) Petri-dishes (9 cm in diameter) and incubated at 27°C. After 4- 5 days incubation period, the developed fungal colonies were purified by hyphal tip technique on PDA medium (200 g Peeled potato, 20 g Dextrose, 20 g Agar; 1000 ml distilled water) at 27°C. The pure fungal isolates were then grown on PDA slants at 27°C and kept in refrigerator at 4°C for further studies. Fungal isolates were
identified by using the morphological features of mycelia as described by Barnett and Hunter (1986).
2.2 Biological Control of Soybean Root Rot by Bioagents
2.2.1 Isolation and identification of the bioagents
For isolation of bio-agents fungi, soil samples were collected from the rhizosphere of healthy alfa alfa plants using dilution plate technique and purified by the single spore method.
The isolated fungi, yeast and bacteria were identified on the basis of their morphological and microscopical characters (Rifai,1969; Nelson, et al. 1983 and Barnett and Hunter 1986). They were identified as Trichoderma sp., Aspergillus sp. and Penicillium sp. according to morphological characteristic. The obtained fungal isolates were grown on PDA slants and kept at 4°C until being used. Yeasts were isolated from samples of the clay soils. The soil adhering to the healthy root systems of alfa alfa plants were washed off thoroughly. Roots were cut from the tip into1-2 cm bits. The root segments were further washed in distilled water and blotted to remove the moisture. One gram of the root material was transferred to 100 ml sterile water in a 250 ml conical flask and shaked for 20 minutes at 250 rpm in a rotary shaker to dislodge bacterial adhering to the root surface. Similarly, one gram of rhizosphere soil was mixed thoroughly in 100 ml sterile water and was processed following serial dilution agar plate technique (Aneja, 2002). Suitable dilutions (10-5 and 10- 6) of both rhizosphere and rhizoplane solutions were placed on King's B medium (King et al., 1954) and nutrient agar medium to isolate fluorescent pseudomonas and total bacteria, respectively. The plates were incubated at (28±2 C) for 24- 48 h. Representative colonies on the NA plates were picked up, purified and preserved in nutrient broth. These strains were kept for following study.
2.2 Evaluation of Antagonistic Activity in Dual Culture Technique in vitro
Petri dishes containing Czapek's agar medium were used, each Petri-dish was divided in two equal halves. The first half was separately inoculated with standard disc of isolates of Fungi isolated from alfa alfa plants rizosphere. The second half was inoculated with an equal disc of R. solani isolate. Each treatment was replicated three times, and inoculated plates with R. solani only were used as control. All Petri-dishes were incubated at 27o C for four days and data was recorded.
Antagonistic percentage was calculated according the following scale index: 0-4, 0= no antagonism; 1= slight antagonism; 2= moderate antagonism; 3= high antagonism and 4=
overgrowth (Hassan, 1992).
2.3 Preliminary Tests for Antagonistic Capability of Bacteria and yeast against Mycelia Growth of the Pathogenic Isolates
The bacterial bioagents were screened under in vitro conditions against R. solani for their antagonistic by using dual culture method as described subsequently (Bouziane et al., 2011). Bacterial bioagents (growing cultures on NA medium) were streaked on the opposite sides of pathogen. Then plates were incubated at 25oC for 7 days. Reduction percentage in liner growth of the tested fungi was determined using the following formula:
R = (C – T/ C) × 100
Whereas R = Percentage of growth reduction C = Diameter of the control hyphal growth T = Diameter of the treated hyphal growth.
Statistical analysis
Data were subjected to statistical analysis using analysis of variance using the Statistical Analysis System, (SAS institute Inc., 1996), and
means were compared using Tuky test according to Gomez and Gomez (1984).
3. Results and Discussion
Effect of certain fungi bioagents against mycelia growth of Rhizocotnia solani under laboratory conditions.
The ability of six isolates of fungi, to inhibit the growth of the root rot fungi Rhizoctonia solani was studied under Lab.
conditions.
Data present in Fig. 1 clearly that the bio- agent fungi exhibited different inhibitory effect against mycelia growth of the tested isolate. In this respect, the highest inhibition of Rhhizoctonia solani growth was occurred by isolate No.1001, followed by 1016 and 1010, which recorded (59.25, 52.76 and 47.16 %), respectively. While, the least inhibition of Rhizoctonia growth was occurred by isolate No.1009, which recorded (31.47%).
These results are in agreement with those obtained by Al-Chaabi and Matrod (2002), Kucuk and Kivanc (2003), Alwathnani and Perveen (2012) and Barari (2016) who reported that T. harzianum caused inhibition of radial colony growth of Rhizoctonia solani in vitro.
Cotxarrera et al. (2002) indicated that the high ability of these fungi to inhibit Rhizoctonia spp.
may return to production of some toxic substances and hydrolytic enzymes like proteases, which could degrade the cell wall of other fungi, mycoparasitsm, competition for nutrient, production of antibiotic, Howell (2003). Coiling their hyphae around the hyphae of this pathogen and parasite on it, or by penetration and subsequent dissolution the FOL cytoplasm, Contreras-Cornejo et al. (2016).
Moreover, Haggag and Mohamed (2007) who reported that T. harzianum mutants produced antifungal metabolites (Trichodermin, gliotoxin and gliovirin) as antibiotics that could reduce the growth rate of F. oxysporum. These results were supported by the work of Yigit and
Dikilitas (2007) which stated that T. harzianum has multi mechanism of actions for controlling plant pathogens that is mycoparasitism via production of chitinase, β 1-3 glucanase, β1-4 glucanase, antibiotic, competition, induced resistance and inactivation of enzyme produced by pathogen in the process of infection.
Effect of certain bacteria bioagents against mycelia growth of Rhizocotnia solani under laboratory conditions.
The ability of eight bacteria isolates to inhibit the growth of the root rot fungi Rhizoctonia solani was studied under laboratory conditions.
Data present in Table 1 and Fig. 2 and 3 clearly showed that the bio-agent bacteria exhibited different inhibitory effect against mycelia growth of Rhizoctonia solani. In this respect, the highest inhibition of R. solani growth was occurred by isolate No. 2002, followed by 2008 and 2004, which recorded (60.5, 47.2and 33.1%), respectively. While, the least inhibition of Rhizoctonia growth was occurred by isolate No. 2007, which recorded (12.95%). These results are consistent with other study (Fatima et al. 2009; Mali and Bodhankar 2009 and Wanted 2012).
The obtained results are in agreement with by Abdelkareem et al. (2014) and Abou-Aly et al. (2015). They reported that many bacteria (PGPR) have potentialities for controlling plant pathogenic fungi such as F. oxysporum in vitro through production of antifungal activities. The high ability of these bacteria to inhibit the Fusarium sp. may return to production of some toxic substances, Antibiotics and hydrolytic enzymes like catechol-type siderophores, chitinase, cellulase and protease, which could degrade the cell wall and were more efficient for inhibition of fungal growth. (El-Mougy et al.
2011).
Table 1. Effect of antagonistic bacteria on mycelial growth inhibition of Rhizoctonia solani in vitro.
Isolates No. Mycelial growth inhibition
2001 30.1 ab
2002 60.5 a
2003 30.6 bc
2004 33.1 bc
2005 18.5 cd
2007 12.95 d
2008 47.2 ab
2010 28.7 de
Control 0 f
Values in the column followed by a similar letter are not significantly different as determined by the LSD test (P < 0.05).
Effect of certain yeast bioagents against mycelia growth of Rhizocotnia solani under laboratory conditions.
The ability of ten isolates of yeast, to inhibit the growth of the root rot fungi Rhizoctonia solani was studied under Lab.
conditions.
Data present in Fig. 3 and 4 clearly that the bio-agent fungi exhibited different inhibitory effect against mycelia growth of the tested isolate. In this respect, the highest inhibition of Rhhizoctonia solani growth was occurred by isolate No.5001, followed by 5007 and 5009, 5002, which recorded (54.56, 449.39, 46.26 and 46.26 %), respectively. While, the least inhibition of Rhizoctonia growth was occurred by isolate No.5006, which recorded (37.93%).
Our results are in close confirmation with the findings of Theradimani et al. (2018) they reported that many yeast have potentialities for controlling plant pathogenic fungi such as R.
soalni in vitro through production of antifungal activities. The high ability of these yeasts to inhibit the Fusarium sp. may return to competition for space and nutrients Filonow, (1998), production of antifungal diffusible metabolites, volatile compounds Payne et al., (2000), production of cell-wall degrading enzymes such as β-1,3- glucanase and mycoparasitism Masih and Paul, (2002).
Fig. 1. Effect of antagonistic fungi on mycelial growth inhibition of Rhizoctonia solani in vitro.
A B C
D E F
Fig. 2. In vitro study, effect of certain bacteria isolates against growth of Rhizoctonia solani. Whereas A: Isolate No 2009, B =No 2002, C 2008, D No 2004; E = 2010 F = 2005 and G is the control.
a
cd
c
ab
bc
a
control 0
20 40 60 80
1001 1004 1009 1010 1014 1016 control
Reduction %
Isolates No.
effect of
Fig. 3. Effect of antagonistic yeast on mycelial growth inhibition of Rhizoctonia solani in vitro.
A B
C
D
Fig. 4. In vitro study, effect of certain yeasts isolates against growth of Rhizoctonia solani. Whereas A: Isolate No 5001, B =No 5001/2, C 5004, and D is the control.
a bc
d cd d d
ab cd
bc d
0 10 20 30 40 50 60
Reduction %
Isolates No.
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رطف ىلع ةيويحلا ةحفاكملا لماوع ضعب ريثأت
Rhizoctonia solani
ربتخملا يف
لع دلاخ ي
ىريسع
، و إ لع دمحم ميهارب ي
مسق ةفاجلا قطانملا ةعارز
، ةيلك لأا ةفاجلا قطانملا ةعارزز ةيئبلاو داصر
، زيزعلادبع كلملا ةعماج
، دج ة سلا ةيبرعلا ةكلمملا ، ةيدوع
صلختسملا .
نع جتانلا ميسربلا روذج نفعت ربتعي
Rhizoctonia solani
يتلا ضارملأا مهأ نم
ةساردلا تفده اذل .لوصحملا جاتنإ يف ةريبك رئاسخ ببستو تاتابنلا نم ديدعلا يف اًيونس ثدحت .هليلقت لقلأا ىلع وأ ضرملا ىلع ةرطيسلل ةيجيتارتسا لضفأ داجيإ ىلإ ةيلاحلا و
هظأ جئاتنلا تر
يجولوفروملا تافصلا مادختساب ةيرطفلا تلازعلا ىلع فرعتلا مت هنأ يرطفلا تايرطفلل ة
لثم ة
R.
solani
. امك ومن طيبثتل ةداضملا ةيويحلا تايرطفلا ضعب ريثأت ةسارد تمت
R. solani
.ربتخملا يف
مقر ةلزعلا يف 2002
ةلوزعملا اهيلت ، 2002
و 2002 ( تلجس يتلاو 00,5
، 24,2 و 33,1
٪ )
ومنل طيبثت ىلعأ ناك ،ددصلا اذه يف يلاوتلا ىلع
R. solani
لاب ناك مقر ةلزع 5001 ةلزعلا اهيلت ،
5004 و 5005 ، 5002 ( تلجس يتلا 52,50
، 225,35 ، 20,20 و 20,20
٪ .يلاوتلا ىلع )
ومن دض ةيريتكب تلالاس ينامث رابتخا مت
R. solani
،ربتخملا يف ذإ
ايريتكبلا عيمج نأ تابثإ مت
ومن ريبك لكشب طبثت ةربتخملا
R. solani
طيبثت ىلعأ ناكو ،ربتخملا يف مقر ةلزعلاب ثدح ومنل
1001 ةلزعلا اهيلت ، 1010
و 1010 ( تلجس يتلا ، 55,25
و 52,40 و 24,10
٪ .يلاوتلا ىلع )
مقر ةلزعلاب ناك رطفلا ومنل طيبثت لقأ امنيب 1005
( تلجس يتلاو 31,24
٪ .) و رشع رابتخا مت
ربتخملا يف ضرمملا لماعلا ومن دض ةريمخلا نم تلالاس
، ةريمخلا عيمج نأ ترهظأو ربتخملا
ة
لوقلا اننكمي ،ماتخلا يف .ربتخملا يف ضرمملا لماعلا ومن ريبك لكشب طبثت إ
نكمي هن مادختسا
لاعف لكشب ضرملا ىلع ةرطيسلل ةئيبلل ةنمأ ةقيرطك ةيويحلا ةحفاكملا لماوع
، صني دقو ح هب
نوعرازملا
، ةيوضعلا ةعارزلا ىف ةصاخو .
