Open Agriculture. 2018; 3: 264–272
Research Article
Irda Safni*, Widya Antastia In vitro antagonism of five rhizobacterial species against Athelia rolfsii collar rot disease in soybean https://doi.org/10.1515/opag-2018-0028 received September 26, 2017; accepted June 26, 2018 1 Introduction
Abstract: Plant Growth Promoting Rhizobacteria Soybean (Glycine max (L.) Merril) is one of the important (PGPR) influence plant growth by a number of direct legume crops worldwide including in Indonesia. National (producing plant growth promoting substances) and demand for soybean increases 18% annually accordance indirect (through prevention of deleterious effects of with domestic population growth, however the soybean phytopathogenic microorganisms) mechanisms. Five production is quite low (Direktorat Pangan dan pertanian species of bacteria were isolated from rhizospheric soils of 2015, Ratulangi 2004). soybean and peanut fields from several locations in North Soybean suffers considerable damage from collar Sumatra. On the basis of morphological and biochemical rot disease incited by Athelia rolfsii (Curzi) (formerly characteristics, the bacteria were identified as Aeromonas Sclerotium rolfsii). A. rolfsii is a soil borne fungal pathogen hydrophila, Burkholderia cepacia, Serratia ficaria, Pantoea which has a wide host range including rice, mungbean, spp. 2, and Vibrio alginolyticus. These species were tested peanut, soybean, sweet potato, banana, wheat and potato in vitro against the causal pathogen of collar rot disease (Agrios 1997). A. rolfsii was first reported as a tomato of soybean, Athelia rolfsii, which is an important soybean blight pathogen in Florida, USA by Rolfs (1892). Saccardo disease in Indonesia. The five species of bacteria were (1911) named the fungal pathogen as Sclerotium rolfsii, subjected to screening of antagonistic activities against which has had taxonomic revision and transferred into A. A. rolfsii in vitro with a dual culture-technique. Of the rolfsii (Tu and Kimbrought 1978). five species, B. cepacia, S. ficaria and V. alginolyticus In Indonesia the yield losses due to collar rot disease were the most effective antagonistic bacteria to control A. affecting soybean and other legumes reach 5-55% in dry rolfsii. B. cepacia, S. ficaria and V. algynolitycus produced land or wet land (Nautiyal 2002, Wahyuningsih 2005). inhibiting zones against A. rolfsii of 98.35%, 97.83% and The disease intensity in the field is mostly more than 5%, 96.97% respectively. All bacterial species showed their which has an impact on the yields economically because it antagonistic activity significantly with the inhibiting causes yield reduction or failure to harvest (Wahyuningsih zone percentage being more than 60%. The experimental 2005). The infection occurs in the early plant growth, results suggested that all bacterial species have a future showing seedling rot or damping-off of seedlings. In potency as a biocontrol agent to reduce A. rolfsii collar rot the older plant (2-3 weeks old), rot symptom occurs in disease of soybean. the collar part of the plant with brown spots and white mycelia on the infected area (Semangun 2000). Keywords: plant growth promoting rhizobacteria, A. rolfsii can be managed by fungicide application, Aeromonas hydrophila, Burkholderia cepacia, Serratia soil solarization, crop rotation and the use of antagonistic ficaria, Pantoea sp 2, Vibrio alginolyticus microorganisms (Punja 1988). To avoid the negative impact of fungicide residue, more enviromental friendly methods such as biological controls have been applied recently (Hardaningsih 2011, Rahayu 2008). These include, the use of beneficial fungi and bacteria (Suryanto 2009). Plant Growth Promoting Rhizobacteria (PGPR), which are a *Corresponding author: Irda Safni, Department of Agrotechnology, group of beneficial bacteria, colonizes rhizosphere, have Faculty of Agriculture, Universitas Sumatera Utara, Medan, 20155, Indonesia, E-mail: [email protected] been shown to have potential to improve plant growth as Widya Antastia, Department of Agrotechnology, Faculty of well as to give plant protection against pathogens direct Agriculture, Universitas Sumatera Utara, Medan, 20155, Indonesia or indirect effects (Saharan and Nehra 2011). This research
Open Access. © 2018 Irda Safni, Widya Antastia, published by De Gruyter. This work is licensed under the Creative Commons Attribution- NonCommercial-NoDerivs 4.0 License. In vitro antagonism of five rhizobacterial species against Athelia rolfsii collar rot disease in soybean 265 was aimed to test the antagonistic effect of rhizobacteria solubilization (Widayanti 2007), Hydrogen Cyanide (HCN) isolated from legumes in North Sumatra against A. rolfsii production (Agustiansyah et al. 2013) and siderophore in vitro. production where the absorbance values were measured by spectrophotometer (Dirmawati 2003). The biochemical assays and the PGPR screening 2 Methods assays were replicated three times.
2.1 Sample collection 2.4 In vitro screening for antagonism Soil samples of soybean’s rhizosphere were collected from districts of Tanjung Selamat, Lubuk Pakam and Binjai, Five selected bbacterial isolates, which were screened North Sumatera, Indonesia, while soil samples of peanut’s based on a PGPR assay in vitro for growth inhibition of rhizosphere were collected from districts of Tanjung the phytopathogenic fungus A. rolfsii. Briefly, 5 μl drops Selamat, Medan Johor and Binjai, North Sumatera. Soil of each bacterial culture (108 CFU/ml) were streaked samples were collected on the basis of random sampling equidistantly on the margins of PDA plates. A mycelial from 5 points from a depth of 5-25 cm between healthy agar plug of 5 mm diameter from a 7-day-old culture of A. plants and infected plants. The soils were composited in rolfsii was grown on PDA plates next to the streak of the one plastic bag before being brought to the laboratory. test bacterium. Control plates not inoculated with bacteria were also prepared. Two independent experiments with each bacterial isolate were performed and each experiment 2.2 Bacterial isolation was replicated three times. Plates were incubated at 28°C for 2 days. Antagonistic activity was assessed by relating Ten grams of soil was mixed with 90 ml of sterile water for mycelia diameter on plates inoculated with bacteria to 15 min. A dilution series of 10-5 was conducted. The soil mycelia diameter on control plates and computing the suspension was streaked onto potato dextrose agar (PDA) percentage of Growth Inhibition (GI %). The inhibition of media, which was used to grow both phytopathogenic the mycelial growth of A. rolfsii was measured by using the fungi A. rolfsii and the bacteria, and incubated for 48 following formula: hours at 28°C. The bacteria grew very well on the PDA media. The homogenous bacterial colonies based on the colony morphology were separated and re-cultured until pure cultures were obtained. where I = inhibition of mycelial growth of the pathogen (%), C = growth of A. rolfsii in the control plate (mm) and T = growth of the pathogen in plates challenged with PGPR 2.3 Bacterial identification (mm) (Kumar et al., 2011)
The bacterial strains were identified on the basis of morphological and biochemical assays for several general 2.5 Statistical Analysis assays to observe the morphological characteristics and biochemical reactions of the bacteria and PGPR screening Statistical analysis was performed using SPSS Statistics assays for determining which strains had potential as version 21.0.0 (International Business Machines PGPR. Corporation, USA). For multiple-group comparisons The morphological assays included colony and cell among the antagonistic bacterial strains and morphologies, while the biochemical assays included phytopathogenic fungi A. rolfsii, two-way ANOVA at P < Gram staining, catalase, oxidase (Schaad et al. 2001), and 0.05 was performed. All data were expressed as means ± physiological assays comprised of growth on different pH SD (standard deviations). values (5.0, 6.0, 7.0, 8.0, 9.0 and 10.0) (Woyesa and Assefa 2011), and utilizing an API® 20E Microbial Identification Ethical approval: The conducted research is not related kit (Biomerieux). The growth at different pH values was to either human or animals use. conducted for the five selected strains. The PGPR screening assays included indole acetate acid (IAA) production (Widayanti 2007), phosphate 266 I. Safni, W. Antastia
3 Results 3.2 Bacterial identification
Colony and cell Morphological characteristics of seventeen 3.1 Bacterial isolation bacterial strains isolated from the rhizospheres of soybeans and peanuts are displayed in Table 2. Colony shapes of the A total of seventeen bacterial strains were isolated from strains were mostly circular with the exception of strain the rhizhosphere of soybeans and peanuts from five PJ23 and PJ22, which had a filamentous shape. The colony locations in North Sumatera, Indonesia (Table 1). Ten colour, margin and elevation as well as cell shape did not strains and seven strains were isolated from soybeans and have much variation (Table 2). Most rhizospheric bacterial peanuts respectively.
Table 1: Bacteria that were isolated from the rhizosphere of soybean and peanut No Bacterial strains Source of Plant rhizhosphere Location
1 SYT51 Soybean Tanjung Selamat 2 SYT22 Soybean Tanjung Selamat 3 SYT21 Soybean Tanjung Selamat 4 SYP21 Soybean Lubuk Pakam 5 SYP42 Soybean Lubuk Pakam 6 SYP43 Soybean Lubuk Pakam 7 SYP31 Soybean Lubuk Pakam 8 SYB43 Soybean Binjai 9 SYB43 Soybean Binjai 10 SYB51 Soybean Binjai 11 PT5 Peanut Tanjung Selamat 12 PT22 Peanut Tanjung Selamat 13 PJ23 Peanut Medan Johor 14 PJ51 Peanut Medan Johor 15 PJ22 Peanut Medan Johor 16 PB3 Peanut Binjai 17 PB42 Peanut Binjai
Table 2: Morphological characteristics of bacterial strains isolated from the rhizosphere of soybean and peanut in North Sumatera, Indonesia Colony morphology Cell morphology
Strains Shape Colour Margin Elevation Cell shape
SYT51 Circular White Undulate Flat Short rods SYT22 Circular White Entire Flat Short rods SYT21 Circular Cream Entire Flat Coccus SYP21 Circular White Undulate Flat Short rods SYP42 Circular White Entire Flat Short rods SYP43 Circular White Undulate Flat Short rods SYP31 Circular White Entire Flat Long rods, in chain SYB43 Circular Cream Entire Flat Short rods SYB42 Circular White Entire Raised Short rods SYB51 Circular Cream Entire Flat Coccus PT5 Circular White Entire Flat Short rods PT22 Circular White Entire Flat Short rods PJ23 Filamentous White Filiform Raised Coccus PJ51 Circular White Undulate Flat Coccus PJ22 Filamentous White Filiform Flat Short rods PB3 Circular White Entire Flat Short rods PB42 Circular White Entire Raised Short rods In vitro antagonism of five rhizobacterial species against Athelia rolfsii collar rot disease in soybean 267 strains were rods and only four strains were cocci (strain 3.3 In vitro screening for antagonism SYT21, SYB51, PJ23 and PJ51). Among the strains, strain PJ22 isolated from peanut and strain SYP21 isolated from In vitro screening antagonism of five PGPR species against soybean were Gram positive and five selected strains A. rolfsii showed that Burkholderia cepacia had the highest could grow well on media with different pHs (Table 3). growth inhibition against A. rolfsii on the second day after The results of the PGPR screening assays are displayed inoculation (98.35%) (Table 5). Serratia ficari and Vibrio in Table 4. On the basis of a PGPR screening assay, 47% alginolyticus also inhibited the growth of A. rolfsii with of the isolates were able to produce IAA, 58.8% could high percentage values (97.83% and 96.97% respectively). solubilize phosphate, none produced HCN and 100% Aeromonas hydrophyla and Pantoea sp. 2 just inhibited produced siderophores. Isolate SYP31 was able to produce the growth of A. rolfsii by 64.83% and 63.13% respectively. IAA in the media without tryptophan addition, while Figure 1 showed the inhibition of five PGPR isolates other isolates did not produce IAA in the media without against the growth of A. rolfsii on PDA media compared tryptophan (Table 4). to the control without the bacterial treatments. All On the basis of PGPR screening assay, 5 best isolates bacterial isolates interfered with the growth of A. rolfsii were selected for further test, i.e. in vitro antagonism because the fungal mycelium could not fill the petri dish. against phytopathogenic A. rolfsii: On microscopic observation, various impacts of PGPR SYP31 isolates on hyphae of A. rolfsii including crooked hyphae SYB43 (A. hydrophyla and V. alginolyticus), shorten hyphae (B. SYT51 cepacia), and lysed hyphae (S. ficaria, Pantoea sp.2, and PB3 V. alginolyticus) (Figure 2) were observed. PB42
These five bacterial isolates were identified using an 4 Discussion and Conclusions API® test kit, and the results were the following: On the basis of production of indole acetic acid (IAA), SYP31 : Aeromonas hydrophila siderophore and solubilization of phosphate, the five SYB43: Burkholderia cepacia selected bacterial strains might have potential to be used SYT51 : Serratia ficaria as PGPR in the field. Bashan and de-Bashan (2005) stated PB3 : Pantoea sp. 2 that mechanisms including N fixation, solubilization of PB42: Vibrio alginolyticus 2
Table 3: Biochemical and physiological assays of bacterial strains isolated from the rhizosphere of soybean and peanut in North Sumatra Strains Gram Oxidase Catalase Growth on different pH
5.0 6.0 7.0 8.0 9.0 10.0
SYT51 Negative + + + + + + + + SYT22 Negative + + NA1 NA NA NA NA NA SYT21 Negative + + NA NA NA NA NA NA SYP21 Positive + + NA NA NA NA NA NA SYP42 Negative + + NA NA NA NA NA NA SYP43 Negative + + NA NA NA NA NA NA SYP31 Negative + + + + + + + + SYB43 Negative + + + + + + + + SYB42 Negative + + NA NA NA NA NA NA SYB51 Negative + + NA NA NA NA NA NA PT5 Negative + + NA NA NA NA NA NA PT22 Negative + + NA NA NA NA NA NA PJ23 Negative + + NA NA NA NA NA NA PJ51 Negative + + NA NA NA NA NA NA PJ22 Positive + + NA NA NA NA NA NA PB3 Negative + + + + + + + + PB42 Negative + + + + + +
Note: 1NA=Not Available 268 I. Safni, W. Antastia
Table 4: Production of indole acetate acid (IAA), HCN, siderophore and phosphate solubilization of rhizospheric bacterial strains Strains IAA1 production Phosphat HCN2 production Siderophore production Solubilization
LB3 + tryptophan LB without Phosphate Media + glisin Media without Media + FeCl3 Media without tryptophan glisin FeCl3
SYT51 + - + - - 1.14 1.65 SYT22 - - + - - 1.99 2.17 SYT21 - - - - - 2.31 2.39 SYP21 - - + - - 2.26 2.31 SYP42 - - - - - 1.50 2.34 SYP43 - - - - - 0.88 1.19 SYP31 + + - - - 1.63 2.20 SYB43 + - + - - 0.61 2.28 SYB42 + - + - - 2.21 2.59 SYB51 - - - - - 1.17 1.17 PT5 - - - - - 0.51 1.78 PT22 + - + - - 0.51 2.04 PJ23 - - + - - 1.88 1.21 PJ51 - - + - - 0.86 2.27 PJ22 + - - - - 2.12 2.63 PB3 + - + - - 2.42 2.47 PB42 + - + - - 1.14 1.77 Note: 1IAA=Indole Acetic Acid; 2HCN=Hydrogen Cyanide; 3LB=Luria-Bertani
Table 5: In vitro screening antagonism of five PGPR species against Athelia rolfsii Treatments Growth inhibition (%) after 2 days inoculation
I II III Total Mean
Aeromonas hydrophila 97.70 96.80 0.00 194.50 64.83ab Burkholderia cepacia 98.10 99.2 98.60 197.70 98.35a Serratia ficaria 97.70 98.40 97.40 293.50 97.83b Pantoea sp. 2 91.00 98.40 0.00 185.40 63.13ab Vibrio alginolyticus 97.70 98.40 94.80 290.90 96.97b phosphate and zinc, sequestration of iron by siderophore a large number of commercially hydrolytic enzymes and production, production of phytohormones such as Auxins, bioactive substances which are beneficial for plant growth cytokinins and gibberellins, production of the enzyme and health (Eberl and Vandamme 2016). B. cepacia is a 1-aminocyclopropane-1-carboxylate (ACC) deaminase ubiquitous soil organism which has been effectively used promote direct plant growth. However, none of the isolates as a biocontrol agent against many plant pathogenic produced hydrogen cyanide. Hydrogen cyanide is toxic fungi such as Colletotrichum gloeosporioides (de Los to some bacteria, fungi, protozoa, mammals and plants, Santos-Villalobos et al. 2012), Pythium-induced damping- but some bacteria utilize hydrogen cyanide as a source of off, Aphanomyces-induced root rot of pea, Rhizoctonia- nitrogen for growth (Knowles 1988). induced root rot of Poinsettia and other fungal diseases Bacteria Burkholderia cepacia, Serratia ficaria and (Parke et al. 1991; King and Parke 1993; Cartwright Vibrio alginolyticus are the best PGPR isolates based on in and Benson 1994; Fridlender et al. 1993). In the USA, vitro screening antagonisms, therefore they are considered several strains of B. cepacia have been registered by The to have potential to control phytopathogen A.rolfsii in the United States Enviromental Protection Agency (EPA) as field. biocontrol agents against plant pathogenic fungi (Eberl Since the first decription of the genus Burkholderia and Vandamme 2016; Rai 2006). The ability of B. cepacia and species of B. cepacia, it has been revealed that this to act as a biocontrol agent is due to its production of species has great potential in biotechnology as it produces various compounds with antifungal activity (Vial et al. Growth inhibition (%) after 2 days inoculation
Treatments I II III Total Mean
Aeromonas hydrophila 97.70 96.80 0.00 194.50 64.83ab
Burkholderia cepacia 98.10 99.2 98.60 197.70 98.35a
Serratia ficaria 97.70 98.40 97.40 293.50 97.83b
Pantoea sp. 2 91.00 98.40 0.00 185.40 63.13ab
Vibrio alginolyticus 97.70 98.40 94.80 290.90 96.97b
In vitro antagonism of five rhizobacterial species against Athelia rolfsii collar rot disease in soybean 269
a b c d e
Aeromonas hydrophila Burkholderia cepacia Serratia ficaria vs Pantoea sp2 vs Vibrio alginolyticus vs A. rolfsii vs A.rolfsii A.rolfsii A.rolfsii vs A.rolfsii
f
A.rolfsii control
Figure 1: In vitro antagonism of Plant Growth Promoting Rhizobacteria isolates against Athelia rolfsii
2007; Schmidt et al. 2009). Additionally, B. cepacia is also chitinases which were evidenced as crucial factors which known to produce antibiotics. Many studies confirmed degraded the cell wall of fungal hyphae (Ordentlich et al. that the inhibitory activity against plant pathogens was 1988). Serratia marcescens also effective killed root-knot associated with the production of several secondary nematode (Meloidogyne sp.) in vitro (Safni et al 2018) metabolites including altericidins, cepacin, and others This study identified Serratia ficaria as the selected PGPR (Kirinuki et al. 1977; Parker et al. 1984; EI-Banna and strains which inhibited growth of A. rolfsii in vitro. There Winkelmann 1998). Although B. cepacia has been proven is no report finding S. ficaria and Vibrio alginolyticus as a as a good biocontrol agent, some strains may pose a biocontrol agent. Grimont et al. (1979) isolated S. ficaria risk to human health which cause severe infections in from fig trees, caprifigs and fig wasps in California and cystic fibriosis (CF) and immunocompromised patients Tunisia and from a small black ant in France. Grimont (Mahenthiralingam et al. 2005; Vandamme and Peeters and Grimont (2006) added that four percent of S. ficaria 2014; Peeters et al. 2013). Because of this reason, the US isolates was isolated from plants. Vibrio species are withdrew the biopesticide product of B. cepacia from the usually found in aquatic environment and sometimes market (https://www.gpo.gov/fdsys/pkg/FR-2004-09-29/ 15a small amount are found in the wood substrates where pdf/04-21695.pdf). However, B. cepacia have been used in earthworms live (Magdoff and Weil 2004). Nursyam (2017) Asia because the bacterial strains of B. cepacia might have studied the primary metabolites of V. alginolitycus isolated different characteristics and function. from sponge Haliclona sp. found they could inhibit the Similarly, the genus Serratia has been widely used growth of the clinical bacterium Staphylococcus aereus in in agriculture as a biocontrol agent against many plant vitro. The results of this study showed that S. ficaria and pathogens. Several selected strains of Serratia plymuthica, V. alginolitycus might have potency as a biocontrol agent, Serratia marcescens and Serratia liquefaciens have been particularly for A. rolfsii. These results suggested both able to reduce plant disease severity using specific PGPR isolates produced IAA and siderophores as well application strategies (Saha et al. 2017). Serratia species as they could solubilized phosphate which help plant produce the red pigments, prodiogiosin and pyrrolnitrin growth and inhibited growth of the phytopathogenic as well as producing chitinase and a siderophore which fungi A. rolfsii. inhibit fungal growth (Saha et al. 2017). Bacteria with This in vitro study revealed that the effectiveness of chitinolytic activity have shown potency as biocontrol A.hydrophila and Pantoea sp. 2 to inhibit the growth of A. agents against plant pathogenic fungi by degrading the rolfsii was not as good as the other three bacterial isolates. cell walls, which are mainly composed of chitin (Someya One replication showed that the bacterial isolates could not et al. 2011). S. marcescens was an effective biocontrol agent inhibit the growth of A. rolfsii. This might occurs because against Sclerotium rolfsii affecting beans and extracellular the bacterial isolates could not compete with the fungal 270 I. Safni, W. Antastia
a b
Aeromonas hydrophila vs Athelia rolfsii Burkholderia cepacia vs Athelia rolfsii
c d
Serratia ficaria vs Athelia rolfsii Pantoea sp.2 vs Athelia rolfsii
e f
16
Vibrio algynolyticus vs Athelia rolfsii normal hyphae of Athelia rolfsii without bacterial treatment
Figure 2: Microscopic observation of the in vitro antagonism of Plant Growth Promoting Rhizobacteria against Athelia rolfsii, a. crooked hyphae, b. shorten hyphae, c. lysed hyphae, d. lysed hyphae, e. lysed and crooked hyphae, f. normal hyphae
17
In vitro antagonism of five rhizobacterial species against Athelia rolfsii collar rot disease in soybean 271
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