INTRODUCTION
One of the important diseases that attack garlic plants is wilt disease caused by the pathogenic fungus Fusarium oxysporum f.sp. cepae. This fungus is categorized as soil-borne pathogens that infect the host through wounds on the root tissue. In recent years there has been an increase of fusarium wilt disease on garlic. High attacks of this disease can reduce garlic yield up to 35-40% (Sintayehu, Sakhuja, Fininsa, & Ahmed, 2011). Garlic is included as an important commodity that is needed by the community. This is because garlic has been mainly used as spices by Indonesian poeple. Most of garlic demands are supplied by import from other countries.
The high value of imports is strongly influenced by the
low productivity of domestic production. One of the domestic garlic productivity is caused Fusarium will attack.
The application of fungicide to control Fusarium wilt has disadvantages towards environment such as chemical residues. These residues was then suspected to have contribution to the occurrence of pathogen resistance and the spread of pathogens on a wider scale. Hence, biological control method becomes an approach for effective control that are more eco-friendly. Biological control method usually known as the use of biological agents. Biological agents are organisms from the group of bacteria, fungi, viruses and other organisms that can be used for the control of pests and disease.
ARTICLE INFO Keywords:
Arbuscula Mycorrhizae
Fusarium oxysporum f.sp cepae Garlic
Lumbu Hijau Article History:
Received: October 23, 2022 Accepted: February 9, 2023
*) Corresponding author:
E-mail: muhammad.syibli@ub.ac.id
ABSTRACT
Wilt disease caused by Fusarium oxysporum is one of the most serious plant diseases in the world. There is no effective contol for. This study investigated the potential of arbuscular mycorrhizal and bacterial antagonists to control F. oxysporum through in vitro and in vivo studies.
In this study, the antagonistic bacteria Stenotrophomonas maltophilia was isolated from mycorrhizal propagation media. Antagonist bacteria S. maltophilia showed antagonistic ability against F. oxysporum with an inhibition zone of 17.9 cm. Antagonistic bacteria and mycorrhizae used in this study significantly reduced the incidence of fusarium wilt in in vivo experiments. It was found that mycorrhizal and S. maltophilia inoculation showed disease incidence rates at 40% and 47.6%. While in the control treatment the incidence of disease reached 90.3%. The biocontrol agents of S. maltophilia and mycorrhizae have a promising prospective strategy to protect garlic plants. These results are expected to provide new insights for sustainable crop protection systems.
ISSN: 0126-0537
Cite this as: Dewi, R. R., Rahmah, S. M., Taruna, A. T., Aini, L. Q., Fernando, I., Abadi, A. L., & Syib’li, M. A. (2023).
The effectiveness comparison between application of indigenous arbuscular mycorrhizal fungal community and stenotrophomonas maltophilia to suppress fusarium wilt incidence on local garlic plant (lumbu hijau). AGRIVITA Journal of Agricultural Science, 45(1), 131-146. http://doi.org/10.17503/agrivita.v45i1.3970
The Effectiveness Comparison Between Application of Indigenous Arbuscular Mycorrhizal Fungal Community and Stenotrophomonas maltophilia to Suppress Fusarium Wilt Incidence on Local Garlic Plant (Lumbu Hijau)
Rifani Rusiana Dewi1), Syarifah Maulidya Rahmah2), Ardiyan Taruna3),Luqman Qurata Aini3), Ito Fernando3),Abdul Latief Abadi3), and Muhammad Akhid Syib’li3)*
1) Graduate Plant Pathology Study Program, Faculty of Agriculture, Universitas Brawijaya, Malang, East Java, Indonesia
2) Agroecotechnology Study Program, Faculty of Agriculture, Universitas Brawijaya, Malang, East Java, Indonesia
3) Department of Plant Pest and Diseases, Faculty of Agriculture, Universitas Brawijaya, Malang, East Java, Indonesia
Arbuscular mycorrhizal fungi (AMF) are symbionts in the plant root by colonizing the tissues in the root cortex. Symbiosis between the plant and the AMF has a mutual beneficial relationship each other.
This fungus has a role in helping growth, increasing the productivity and quality of crops, especially on marginal lands that are less fertile. AMF also has the ability to increase macro- and micronutrients and can absorb nutrients in a bound form and is not available for plants such as phosphorus. This fungi also has the ability to protect plant root from soil borne pathogen (Goicoechea, 2020).
Antagonistic bacteria are one of the biological control agents that produce a compound that can be used to control pathogens that cause plant diseases.
Antagonistic bacteria can also increase plant growth through several mechanisms such as providing plant protection from plant pathogen attacks. The presence of this antagonistic bacteria can reduce damage due to plant pathogens. Some antagonistic bacteria have the ability to control several soil-borne pathogens, both in vitro and in vivo. The inhibition of antagonistic bacteria against pathogens through several mechanisms such as antibiosis, competition or promoting plant growth (Alabouvette, Olivain, & Steinberg, 2006).
Biological agents such as antagonistic bacteria and arbuscular mycorrhizal are potential agents to control wilt disease caused by Fusarium oxysporum in some plants. Based on previous study conducted by Rajamohan et al. (2019) the application of AMF (Glomus mosseae) as much as 50 g/kg of soil can reduce the incidence of Fusarium wilt disease to 15.62% compared to that without mycorrhiza which reaches 45.87% in onion plants. Whilst, bacterial species are antagonistic to F. oxysporum are Pseudomonas, Streptomyces, and Bacillus. Based on in vitro study, it was found that the antagonistic bacteria can inhibit the F. oxysporum with an inhibition zone between 4.09 – 74.97%. However, in previous study, there is limited information about the effectiveness comparison between the AMF and antagonistic bacteria against fusarium wilt disease on
garlic plants. The aim of this research is to evaluate the effectiveness of AMF and antagonistic bacteria to control Fusarium wilt disease of garlic plants.
MATERIALS AND METHODS
The research was conducted from October 2021 to April 2022, covering laboratory and greenhouse work. The experiment was conducted at the Laboratory of Biological Control and Greenhouse, Department of Plant Pests and Diseases, Faculty of Agriculture, Universitas Brawijaya.
Fungal and Bacterial Isolate
The F. oxysporum was isolated from Allium plant which showed disease symptoms, cultured with PDA medium and stored at -200C based on filter paper stock method. The filter paper was autoclaved for 15 minutes at 1210C. The sterile filter paper was placed in a petri dish containing PDA. Then F. oxysporum was taken using ose needle and incubated. After fungal colonies grew, the filter paper was taken and let them to dry. Furthermore, the filter paper was stored in a sterile container and stored at a temperature of -200C (Duque, 2016). Antagonistic bacteria was isolated from culture medium of AMF, cultured with NA medium and stored at -200C with glycerol 40%.
Morphological Identification of Fungi
Identification of F. oxysporum based on their macroscopic and microscopic characteristics.
Macroscopic observations was made by culture the F.
oxysporum on PDA medium. After the isolates were incubated for 7 days, their shape and color of colony were observed. While the microscopic characteristics were observed by slide culture method. Slide culture was made by cutting the PDA media and then placing it on the object glass and closing it with a cover glass (Fig. 1). The object glass was put in a moist petridish then incubated for 2-3 days. Observations were made using a scanning electron microscope (SEM: Hitachi TM3000 SM) at the Bioscience Laboratory, Universitas Brawijaya.
Fig. 1. Slide culture technique
Molecular Identification of Bacteria
The identification of bacterial was carried out based on the results of 16S-RNA gene sequencing by the PT Genetika Science Indonesia. 16S-RNA gene sequencing consisted of DNA extraction, 16S-RNA gene amplification by PCR, purification of PCR products and sequencing. The universal primers used to amplify DNA using PCR were 27F (5’-AGAGTTTGATCMTGGCTCAG–3’) and 142R (5’– GGTTACCTTGTTACGACTT– 3’). Initial denaturation stage of 95°C for 3 minutes. The proccess of denaturation at 95°C was 15 seconds, annealing of 52°C was 30 seconds, extension of 72°C was 45 seconds with each step was repeated for 35 cycles. Then for the final extension at 72°C was 3 minutes and continued with hold at 4°C. The result of genome amplification were electrophoresed in agarose and sequenced. To identify, the sequences data obtained were compared with nucleotide sequences of NCBI using BLAST on the website http://www.ncbi.nlm.gov/BLAST. The phylogenetic tree was constructed using NCBI using the neighbor- joining method.
Arbuscular Mychorrhizal Propagule Test Value Arbuscular mycorrhizal propagules were calculated using marigold roots as an indicator.
Arbuscular mycorrhizal dilutions were carried out in a series of 50, 5-1, 5-2, 5-3 until 5-10 ml. After 5 weeks after planting, the roots of marigold were taken and the roots were stained. The steps were taken from the root staining method adapted from Koske & Gemma (1989). The roots are washed with running water until there is no remaining sand then the roots are cut for about 2 cm. The roots were then added with 10% KOH and heated for about 10-15 minutes. Then the roots were soaked in 1%
HCl for 3 minutes and rinsed thoroughly. Then the roots were soaked in a 0.05% lactofenol blue and heated for 10-15 minutes. The stained roots were then observed using a microscope. Infected roots are given a positive sign, while non-infected roots are given a negative sign. All of these data were then processed using the following formula 1 for calculating propagules (Alexander, 1965).
MPN/g = (∑ gj) / (∑ tjmj ∑ (tj-gj)mj) (½)...1) Where: MPN/g = Most Probable Number; ∑ gj = number of positive tray at selected dilution; ∑ tjmj = amount of sample in all tray at the selected dilution; ∑ (tj-gj)mj = amount of the sample in the negative tray at the selected dilution.
Arbuscular Mycorrhizal Diversity
Genomic DNA extraction from soil sample was carried out using ZymoBIOMICS DNA Miniprep Kit (Zymo Research, D4300). DNA concentration was determined using both NanoDrop spectrophotometers and Qubit fluorometer. Library preparations were conducted using Kits from Oxford Nanopore Technology.
Nanopore sequencing was operated by MinKNOW software version 22.05.7. Basecalling was performed using Guppy version 6.1.5 with high-accuracy model. The quality of FASTQ files were visualized using NanoPlot, and quality filtering was performed using NanoFilt. Filtered reads were classified using Centrifuge classifier. Fungi index was built using NCBI ITS RefSeq database (https://ftp.ncbi.nlm.nih.gov/refseq/TargetedLoci/).
Downstream analysis and visualizations were performed using Pavian (https://github.com/
fbreitwieser/pavian), Krona Tools was adapted from Ondov, Bergman, & Phillippy (2011) (https://github.
com/marbl/Krona), and RStudio using R version 4.2.0 (https://www.R-project.org/).
In Vitro Assays
The bacteria tested for antagonists with F.
oxysporum was selected based on the inhbition to the the growth of pathogens. The pathogen F. oxysporum is placed in the central part of the NA medium, then the isolate of bacterial isolates were suppressed on the right, left, upper, lower sides of the pathogen F. oxysporum. Observation was carried out for 5 days after inoculation. The observed parameter was the radius of the pathogen F. oxysporum against the colony of bacteria. Besides that, the calculation of inhibitory growth percentage was carried out using the equation 2.
Where: IH = inhibitory growth percentage; R1 = the radius of the pathogenic fungi colony away from the antagonist fungal colony; R2 = the radius of the pathogenic fungus colony come up the antagonist fungal colony.
In Vivo Assays
F. oxysporum that has been culture in PDA media was propagated using corn rice media.
Sterile corn rice then inoculated with F. oxysporum in LAFC. After inoculated, corn rice was incubated until F. oxysporum grew in the corn rice for approximately 14 days. The planting medium used
...2)
Table 1. Experimental treatments
Treatment Description
P0 F. oxysporum
P1 F. oxysporum + 20 gram mycorrhiza P3 F. oxysporum + 30 ml antagonistic bacteria is a mixture of horticultural soil: red soil: sand (v: v:
v = 1:1:1). The planting medium was sterilized by adding 4% formalin. Inoculation of the pathogenic fungi F. oxysporum was carried out by homogenizing the planting medium. The planting media that already contains F. oxysporum was then given the treatments. The treatments are 20 grams arbuscula mycorrhiza, 30 ml antagonistic bacteria, and control (Table 1).
and microscopically using a scanning electron microscope.
Based on macroscopic observation, the fungus has white colonies and filaments like cotton.
While, when they were observed from lower surface, the fungi colonies also had a pink or purple pigment (Fig. 2). This is in line with the characteristics of F.
oxysporum described by Trabelsi et al. (2017). F.
oxysporum has a white mycelium and a purplish undersurface. In addition, when the colony had been too old the fungi will turn into brownish yellow from the center and continue to the edge. This is in accordance with Leslie & Summerell (2007) that stated when the colony of F. oxysporum was too old, the colony would change into yellow or orange.
The texture of surface colony is smooth like wool with flat edges. This pathogen has dense and thick mycelia. The shape of the colony was rounded with concentric growth as seen in daily growth observations. The time required for F. oxysporum to fill the petri dish (d = 9 cm) on PDA media was 7 days.
Microscopic observation was carried out using a scanning electron microscope. In general, Fusarium produce three types of vegetative spores namely macroconidia, microconidia and chlamyodospores (Kalman et al., 2020)there have been accumulating reports from farmers and field extension personnel on the increasing incidence and spread of onion (Allium cepa. Based on microscopic observations, this isolate was identified as F. oxysporum. This is indicated by the presence of macroconidia measured 32-36 μm long and 3 μm wide (Fig. 2). These results confirmed the study on the morphology of F. oxysporum by Ciampi et al. (2009) which stated that the macroconidia of F.
oxysporum had a length range of 27-55 μm and a width of 3-5 μm. It can be seen that macroconidia had spherical shape, 2-3 septa and hyaline. This statement also explained by Fourie, Steenkamp, Ploetz, Gordon, & Viljoen (2011) that F. oxysporum was characterized by the presence of macroconidia formed from monophialides with three septa.
Furthermore, measurement of the incidence of disease was conducted at 2 WAP to 4 WAP.
The disease incidence was measured by dividing the number of affected plants by the total plant population and then multiplied by 100% (formula 3).
Where: I = intensity/severity of damage/attack (%);
n = number of dead plants; N = total number of observed plants.
Statistical Analysis
All data were analyzed using T test with a significant level 5%. Data analysis was performed using R studio software. The data that was analyzed was then presented in the form of tables and boxplotsy.
RESULTS AND DISCUSSION Fusarium Characterization
The pathogenic fungi Fusarium spp. have known to have many morphologically different species. Characterization of F. oxysporum was carried out macroscopically on PDA media
...3)
Remarks: (A) = colony upper surface, (B) = colony lower surface, (C) = conidia, (D) intact conidia, (E, F) mycelium Fig. 2. Morphology characterization of F. oxysporum
Morphology Characterization of Bacteria
The characteristics of B8 was confried by the obervation on the bacterial morphology. Microscopic observations were carried out based on several characteristics like color, shape, edge, size and elevation angle. The morphological characteristics of B8 bacteria can be seen in the Fig. 3.
Bacterial colonies were grown on nutrient agar media. Microscopic observations using a microscope with a magnification 20 X. Isolate B8 had the characteristics of circular colonies, yellowish cream, convex elevation, smooth and shiny colony surface and did not produce mucus. Colonies of B8 bacteria are small about 2.8 mm with flat edge (Fig.
3).
Molecular Identification of Bacteria
The bacteria was identified molecularly to determine to species level. Antagonistic bacterial isolates were identified using universal 16S RNA primers. The PCR products then were sequenced to determine the nucleotide sequence. Previously, to determine the success of the PCR product, electrophoresis was carried out using agarose gel.
Electrophoresis results of antagonist bacteria showed that the molecular weight of the isolates was around 1500 bp (Fig. 4). The species identification was carried out by sequencing the PCR products.
The results of the sequencing were then confirmed to the Basic Local Alignment Search Tool (BLAST) program to determine the homology of similar species.
Phylogenetic tree was created to analyze the close relationship between isolates. One method that is often used was neighbor-joining method.
The results of the phylogenetic tree showed that the bacterial isolate B8 was closely related to
Stenotrophomonas maltophilia strain 2013-SM24 (Fig. 5). In addition, the results of the BLAST analysis showed that the bacterial sequence B8 had a similar base arrangement to the isolate Stenotrophomonas maltophilia (accession number: CP040436.1) with a similarity level of 99%. According to Hofstetter, Buyck, Eyssartier, Schnee, & Gindro (2019)the number of ITS sequences associated to the wrong taxon name appears to be around 30%, even higher than previously estimated. Such results rely on the in-depth re-examination of BLAST results for the most interesting species that were collected, viz. first records for Switzerland, rare or patrimonial species and problematic species (when BLAST top scores were equally high for different species if the similarity reaches 99%, it indicates the possibility of species.
Thus, the bacteria is confirmed Stenotrophomonas maltophilia.
In general, the identification was conducted both of morphologically and molecularly. The morphological method was the initial stage of identification through observation. Meanwhile, molecular identification was designed to improve the accuracy and confirm the results of morphological identification. The results of morphological observations showed that the colonies of Stenotrophomonas maltophilia were yellow with a circular shape (Fig. 3). These results in line with the statement by Ghosh, Chatterjee, & Mandal (2020) that most of the colonies of S. maltophilia have yellow color. The same thing was also explained by Mahdi, Eklund, & Fisher (2014) that S. maltophilia had small circular colonies with a gray to yellow color.
Thus, the results of morphological and molecular identification showed that the B8 bacteria closely related to species Stenotrophomonas maltophilia.
Remarks: (A) = B8 macroscopic, (B) = B8 microscopic Fig. 3. Morphological characterization of B8 bacteria
Arbuscular Mycorrhiza Diversity
The observation of mycorrhizae diversity with the next generation sequencing platform Nanophore indicates that the mycorrhizae was belong to the class Glomeromycetes. The mycorrhizae consist of some genus like Diversispora, Acaulospora and others. Each genus has a different percentage distribution (Fig. 6). From the total mycorrhizae found, most were dominated by the genus
Diversispora with a percentage of 46% consisting of Diversispora aurantia and Diversispora spurca each with 23%. These was then followed by the genus Acaulospora with 18% consisting of Acaulospora entreriana (9%), Acaulospora cavermataI (7%) and Acaulospora leavis (2%). In addition, 14% of the Dentiscutata savannicola species were also found and the remaining 20% were included in other Glomeromycetes.
Fig. 4. Visualization PCR result of B8 bacteria
Fig. 5. Phylogenetic construction of B8 bacteria using the neighbor-joining method
The high diversity of Glomeromycota obtained is in accordance with the statement of Davison et al.
(2015) which states that Glomeromycetes taxa can be found in all continents, but the some diversities was found in the tropics. This is in accordance with the geographical conditions of Indonesia which is a tropical area. In addition, in the class Glomeromycetes, the genus Acaulospora were detected. The results of the study by Marinho, Da Silva, Oehl, & Maia (2018) stated that Acaulospora has a large number and the diversity is most often
found in in tropical rain forests. However mycorrhizal diversity may vary at each location. According to da Silva, de Souza, da Silva, Oehl, & Maia (2017) the characteristics of the environment such as climate and edaphic factors can affect the distribution and diversity of mycorrhizae. In their natural habitat, the distribution of Glomeromycetes is influenced by several other factors such as soil properties, climate, and vegetation (Pereira, da Silva, Goto, Rosendahl, & Maia, 2018).
Fig. 6. Diversity of mycorrhizal fungal community detected
Arbuscular Mycorrhizal Propagule Test Value One indicator to observe the presence of mycorrhizal infection is by root staining. The results of the root staining found that there was infection from vesicles from arbuscular mycorrhizae which were round and oval in shape. The process of mycorrhizal arbuscular infection begins with the formation of an appressorium on the root surface.
Appressorium then enters the epidermis and forms intracellular hyphae. These internal hyphae will develop to form arbuscules and vesicles. The root staining process was carried out, and made observations using a microscope, which can be seen in Fig. 7.
Based on microscopic observations, it shows that there are several parts of the mycorrhizal structure. A bulging and spherical structure was found (Fig. 7. A. B. E. F) called vesicular. According to the explanation of Islam et al. (2022), vesicles are usually oval, and some are spherical. Vesicular is a fungal structure originating from swelling of internal hyphae. Vesicular has a thick wall and contains a lot of lipid, so it serves as food storage (Maiti &
Ghosh, 2020)such as plant growth medium, level of contamination, environmental conditions, restoration practices, age of reclamation, surrounding flora and
fauna community, selection of plant species, and microbial community. Soil characteristics effect plant growth and community development. The selection of plant species is used as an important tool to accelerate the pedogenesis process and initiate progressive succession. Plant–soil interactions can be used as major tools for restoration (e.g., ameliorate soil properties favoring autochthonous species. In addition, microscopic observations also showed the presence of long structures called hyphae (Fig. 7. C. D). These hyphae can increase the area of absorption (Bowles, Barrios- Masias, Carlisle, Cavagnaro, & Jackson, 2016). So that it can absorb minerals from the soil that are not accessible to plant roots. Furthermore, each root infection from each dilution was calculated to determine MPN values. From these observations, the results are as presented in the Table 2.
The data is then calculated into the MPN (Most Probable Number) formula. MPN method can calculate the number of infective propagules and estimate the population. From these data, the MPN value was 5.490 x 103, meaning that the soil contained 5,490 x 103 infective propagules per gram of soil.
Fig. 7. Mycorrhizal infection in plant roots
Antagonism Test
The percentage of antagonist inhibitory was tested to observe the development of the inhibitory
zone. Fig. 8 showed comparison diameter of the pathogen F. oxysporum with S. maltophilia and control on the first to fifth day after inoculation.
Table 2. MPN calculation
Dilution U1 U2 U3 U4 U5 (+) (-)
50 + + + + + 5/5 0/5
5-1 + + + + + 5/5 0/5
5-2 + + + + + 5/5 0/5
5-3 + + - + + 4/5 1/5
5-4 + + - + + 4/5 1/5
5-5 + + + + - 4/5 1/5
5-6 - + - + + 3/5 2/5
5-7 - + + - + 3/5 2/5
5-8 + - - - - 1/5 4/5
5-9 - - - - - 0/5 5/5
5-10 - - - - - 0/5 5/5
MPN 5.490,745 propagul/gram
Remarks: (A) 1 DAI (B) 2 DAI (C) 3 DAI (D) 4 DAI (E) 5 DAI
Fig. 8. Boxplots showing the diameter inhibition measured during the antagonism test
E C
Remarks: (A) 1 DAI (B) 2 DAI (C) 3 DAI (D) 4 DAI (E) 5 DAI
E C
The application of antagonistic bacteria B8 has a significant effect (P<0.05) based on the diameter inhibition from first to fifth day after inoculation, in the treatment of S. maltophilia, the diameter colonies of F. oxysporum was inhibited (Fig. 8). On the fifth day the average diameter of colonies given S. maltophilia has a smaller diameter of up to 1.79 cm. While the average colony diameter in the control treatment was 3.25 cm. So that when calculated, the percentage of inhibition of S. maltophilia is 45.07%. The existence of this inhibition process can be seen from the formation of a clear zone in the S. maltophilia treatment (Fig. 9).
Based on Fig. 9, it can be seen that the antagonistic bacteria S. maltophilia produce clear zones. According to Ranjbariyan, Shams- Ghahfarokhi, Kalantari, & Razzaghi-Abyaneh (2011) the presence of antagonism activity was seen by the presence of a clear zone as a form of inhibition of fungal growth. The presence of a clear zone indicates the ability of antagonistic bacteria to inhibit the activity of the pathogenic F. oxysporum. This is in accordance with the research by Jankiewicz, Brzezinska, & Saks (2012) that Stenotrophomonas maltophilia had antagonistic activity against fungal pathogens such as Fusarium, Rhizoctonia and Alternaria through the ability to synthesize chitinase.
The clear zone produced by antagonistic bacteria against F. oxysporum indicated that
these bacteria produced antibiotics. According to Haggag & Mohamed (2007), the antibiosis activity was determined by the presence of an inhibition zone characterized by a clear zone. Antibiosis is the ability of antagonistic organisms to inhibit the growth of pathogens through several mechanisms such as the production of secondary metabolites, siderophores, several enzymes or other toxic substances.
The genus Stenotrophomonas can be a biological control because it can produce several metabolites and antifungal compounds. Some antifungal compounds that have been identified are Maltophyllin (Jakobi et al., 1996) and xanthobaccin (Nakayama, Homma, Hashidoko, Mizutani, &
Tahara, 1999). The antifungal activity was also initiated by the production of volatile organic compounds (VOCs). According to Kai, Effmert, Berg, & Piechulla (2007) that volatile organic compounds will affect pathogens by inhibiting the growth of fungal mycelium. In addition, S.
maltophilia produces enzymes that also play a role in controlling fungal pathogens such as chitinase and proteases. Chitinase has the ability to lyse the cell wall of pathogenic fungi thus it can protect plants. The antifungal activity by S. maltophilia was a combination of several mechanisms, either the secretion of enzymes or antifungal metabolites.
Remarks: (A) control (B) Stenotrophomonas maltophilia
Fig. 9. Antagonism test developed by S. maltophilia against F. oxysporum
Diseases Incidence
Disease incidence assays were carried out in vivo. It’s to determine the effect of S. maltophilia bacteria and arbuscular mycorrhizae related to the inhibiting the fusarium wilt disease in garlic plants.
Observations were made for four weeks after inoculation (WAI). The result is shown in Table 3.
media to be more resistant to the development of pathogens (Fig. 10 B).
The treatment with S. maltophilia also showed a significant difference in the third week of observation. In the control treatment, the incidence of the disease reached 90%. By giving S.
maltophilia bacteria, the incidence of disease can be reduced to 47.5% (Table 4). This proves that these bacteria have antagonistic properties that can inhibit the incidence of fusarium wilt disease until 42.5%. The ability of these bacteria to suppress disease incidence can be caused by their ability to induce systemic resistance of plants to activate plant resistance genes and from their ability to produce antimicrobial compounds that play a role in inhibiting and suppressing the growth of the pathogen F. oxysporum (Fig. 10 A)
Table 3. The percentage of disease incidence fusarium wilt disease between control and mycorrhiza
Treatment 2 WAI 3 WAI 4 WAI
Control 30 80.8 90
20 gram mycorrhiza 15* 30.83 * 39.16 * Table 3 showed a significant difference compared to the control treatment. The application of mycorrhizae can reduce the incidence of disease, which initially reached 90% in the control treatment, and 39.16% when given mycorrhizal treatment. In other words, the application of mycorrhizal was able to reduce the incidence of disease by 50.84% when compared to the control treatment. This occurs because mycorrhizae has affected the growing
Table 4. The percentage of disease incidence fusarium wilt disease between control and antagonism bacteria
Treatment 2 WAP 3 WAP 4 WAP
Control 30 80.8 90
30 ml bacteria 24.16 34.16 * 47.5 *
Remarks: (A) S. maltophilia (B) mycorrhiza (C) control
Fig. 10. Comparison of disease incidence from several treatments
The application of arbuscular mycorrhizae or S. maltophilia bacteria was able to inhibit the suppression of fusarium wilt disease. The development of Fusarium pathogens can be suppressed by biocontrol agents if there has been a symbiosis with the host plants. The reduced incidence of disease in garlic treated with mycorrhizae was associated with several mycorrhizal mechanisms, such as increased nutritional status of plants, changes in root morphology, and the change in microbial community.
Morphological changes experienced by plants treated with mycorrhizae related with the lignification activity. According to Parihar, Rakshit, Rana, Tiwari, & Jatav (2020) the presence of mycorrhizae will stimulate the formation of lignin in the root endodermis. One of the important factors for plant resistance response is the thickness and strength of the cell walls of the epidermis and endodermis. It is becasue these parts are the outermost tissues as a place of penetration of pathogens. So that by thickening this tissue it will be a barrier against pathogen penetration and make the plant more resistant (Boutaj, Meddich, Roche, Mouzeyar, & El Modafar, 2022).
There are several indirect mycorrhizal mechanisms in suppressing pathogens, including through increasing plant nutrient uptake and directly through the production of compounds that work against pathogenic organisms. The increase in plant nutrient uptake is related to the presence of hyphae which can expand the area of root absorption. The presence of external hyphae in the rhizosphere can increase nutrient uptake. Plants with good nutrient status have low sensitivity to pathogens. Meanwhile, mycorrhizae is able to produce bioprotectants against fungi of the genus Fusarium (Koltai & Kapulnik, 2010).
The application of S. maltophilia bacteria was not only effective on a laboratory scale, but also able to reduce the incidence of garlic disease under in vivo test. The genus Stenotrophomonas has the ability as a biological control because they are able to produce several metabolites and antifungal compounds (Alijani, Amini, Ashengroph, &
Bahramnejad, 2020). Some antifungal metabolites produced by S. maltophilia were Maltophilin and Xanthobaccin. In addition, this species also produces volatile organic compounds (VOC) and several enzymes such as chitinase, protease, glucanase. These enzymes were able to affect the
pathogen by inhibiting the germination of conidia (Ghosh, Chatterjee, & Mandal, 2020). It can be seen that there was a decrease in the incidence of disease in the bacterial treatment (Table 4). The same result was also obtained by John & Thangavel (2017) that by giving bacteria S. maltophilia the growth inhibition of pathogen reached 66.6%.
In addition, the application of mycorrhizae and S. maltophilia antagonist bacteria is also associated with changes in the microbial community. The results of previous studies reported that there was an increase in microbes after 20 weeks of mycorrhizal application (Ladygina et al., 2010). Mycorrhizae will stimulate the abundance of bacteria, actinomycetes, saprophytic fungi, and other mycorrhizae. This positive interaction between mycorrhizae and microbes is made possible by the presence of mycorrhizae that can supply carbon from plants (Nottingham et al., 2013). Carbon is a source of nutrition for other free-living microorganisms. It is also explained by Barea, Pozo, Azcón, & Azcón-Aguilar (2005) that mycorrhizal symbiosis will produce mycelium. This mycelium is used as a food source thus creating an ideal environment for microbial activity. Changes in the microbial community will cause the stimulation of microbiota that may be antagonistic to root pathogens. This is in accordance with the study by Zhou et al. (2020)adding a further dimension of complexity. The plant-AM fungus-bacterium system is considered a continuum, with the bacteria colonizing not only the plant roots, but also the associated mycorrhizal hyphal network, known as the hyphosphere microbiome. Plant roots are usually colonized by different AM fungal species which form an independent phosphorus uptake pathway from the root pathway, i.e., the mycorrhizal pathway. Plant roots are usually colonized by various arbuscular mycorrhizal (AM that the application of Glomus fasciculatum can significantly inhibit the development of Phytium aphanidermatum. In addition, application of S. maltophilia also promotes the recruitment of other beneficial microbes. With the formation of a more diverse microbial community, there is a tendency for more complex interactions (Gao et al., 2021). This microbial diversity will trigger changes in a microbiome. The microbiome network consists of various components that are interconnected and interact with each other either mutualistic, synergistic, commensalistic, ammensalistic or parasitic (van der Heijden &
Hartmann, 2016). Through this interaction, it will potentially affect each component for either soil fertility or plant health. Previous research by Agler et al. (2016) showed that interrelated species in a microbiome can influence pathogen inhibition. This happens because there is a possibility that the relationship will stimulate the presence of beneficial organisms or prevent entry of pathogens.
Sometimes the use of biocontrol agents is not always effective in controlling plant pathogens.
In other words, the use of biocontrol agents carried out in the laboratory is not necessarily effective when applied in the field. This happens because of the complex interactions among pathogens, hosts, biocontrol agents and environmental conditions. This is in accordance with the statement by Goicoechea (2020) that the effectiveness of biocontrol agents is influenced by environmental conditions such as temperature, soil and water.
In addition, this ineffectiveness is sometimes also influenced by a decrease in the ability of biocontrol agents to adapt to biotic and abiotic environments.
Therefore, in order to be successful, very important to know the application of biocontrol agents both of mycorrhiza or antagonism at the right time.
CONCLUSION
This study used biocontrol agents in the form of arbuscular mycorrhizae and the antagonist bacterium S. maltophilia to suppress Fusarium wilt disease. The results showed that the arbuscular mycorrhizal fungus and the antagonistic bacterium S. maltophilia were strong enough against the pathogen F. oxysporum that causes wilt disease in garlic. The application of arbuscular mycorrhizae and S. maltophilia individually was able to reduce the percentage of disease incidence. These results provide a promising strategy in providing protection against garlic plants. Therefore, the future studies would be essential to investigate the combined application of arbuscular mycorrhizal fungal community and S. maltophilia to suppress Fusarium wilt.
REFERENCES
Agler, M. T., Ruhe, J., Kroll, S., Morhenn, C., Kim, S.-T., Weigel, D., & Kemen, E. M. (2016). Microbial hub taxa link host and abiotic factors to plant microbiome variation. PLoS Biology, 14(1), e1002352. https://doi.org/10.1371/journal.
pbio.1002352
Alabouvette, C., Olivain, C., & Steinberg, C. (2006).
Biological control of plant diseases: The European situation. European Journal of Plant Pathology, 114(3), 329–341. https://doi.
org/10.1007/s10658-005-0233-0
Alexander, M. (1965). Most-probable-number method for microbial populations. In A. G. Norman (Ed.), Methods of Soil Analysis, Part 2: Chemical and Microbiological Properties (pp. 1467–1472).
American Society of Agronomy, Inc. https://doi.
org/10.2134/agronmonogr9.2.c49
Alijani, Z., Amini, J., Ashengroph, M., & Bahramnejad, B.
(2020). Volatile compounds mediated effects of Stenotrophomonas maltophilia strain UN1512 in plant growth promotion and its potential for the biocontrol of Colletotrichum nymphaeae.
Physiological and Molecular Plant Pathology, 112, 101555. https://doi.org/10.1016/j.
pmpp.2020.101555
Barea, J.-M., Pozo, M. J., Azcón, R., & Azcón-Aguilar, C.
(2005). Microbial co-operation in the rhizosphere.
Journal of Experimental Botany, 56(417), 1761–
1778. https://doi.org/10.1093/jxb/eri197
Boutaj, H., Meddich, A., Roche, J., Mouzeyar, S., & El Modafar, C. (2022). The effects of mycorrhizal fungi on vascular wilt diseases. Crop Protection, 155, 105938. https://doi.org/10.1016/j.
cropro.2022.105938
Bowles, T. M., Barrios-Masias, F. H., Carlisle, E. A., Cavagnaro, T. R., & Jackson, L. E. (2016).
Effects of arbuscular mycorrhizae on tomato yield, nutrient uptake, water relations, and soil carbon dynamics under deficit irrigation in field conditions. Science of The Total Environment, 566–567, 1223–1234. https://doi.org/10.1016/j.
scitotenv.2016.05.178
Ciampi, P., L., Nissen, M., J., Venegas, G., E., Fuentes, P., R., Costa, L., M., Schöbitz, T., R., … Alvarado, A., P. (2009). Identification of two species of Fusarium link that cause wilting of colored callas (Zantedeschia aethiopica (L.) Spreng.) cultivated under greenhouse conditions in Chile. Chilean Journal of Agricultural Research, 69(4), 516–525. https://doi.org/10.4067/S0718- 58392009000400006
da Silva, I. R., de Souza, F. A., da Silva, D. K. A., Oehl, F., & Maia, L. C. (2017). Patterns of arbuscular mycorrhizal fungal distribution on mainland and island sandy coastal plain ecosystems in Brazil.
Microbial Ecology, 74(3), 654–669. https://doi.
org/10.1007/s00248-017-0979-x
Davison, J., Moora, M., Öpik, M., Adholeya, A., Ainsaar, L., Bâ, A., … Zobel, M. (2015). Global assessment of arbuscular mycorrhizal fungus diversity reveals very low endemism. Science, 349(6251), 970–973. https://doi.org/10.1126/
science.aab1161
Duque, P. (2016). Environmental responses in plants:
Methods and protocols. Methods in Molecular Biology. New York: Humana. https://doi.
org/10.1007/978-1-4939-3356-3
Fourie, G., Steenkamp, E. T., Ploetz, R. C., Gordon, T.
R., & Viljoen, A. (2011). Current status of the taxonomic position of Fusarium oxysporum formae specialis cubense within the Fusarium oxysporum complex. Infection, Genetics and Evolution, 11(3), 533–542. https://doi.
org/10.1016/j.meegid.2011.01.012
Gao, M., Xiong, C., Gao, C., Tsui, C. K. M., Wang, M.- M., Zhou, X., … Cai, L. (2021). Disease-induced changes in plant microbiome assembly and functional adaptation. Microbiome, 9(1), 187.
https://doi.org/10.1186/s40168-021-01138-2 Ghosh, R., Chatterjee, S., & Mandal, N. C. (2020).
Stenotrophomonas. In Beneficial Microbes in Agro-Ecology: Bacteria and Fungi (pp. 427–
442). Academic Press. https://doi.org/10.1016/
B978-0-12-823414-3.00020-4
Goicoechea, N. (2020). Mycorrhizal fungi as bioprotectors of crops against Verticillium Wilt—A hypothetical scenario under changing environmental conditions. Plants, 9(11), 1468. https://doi.
org/10.3390/plants9111468
Haggag, W. M., & Mohamed, H. A.-L. A. (2007).
Biotechnological aspects of microorganisms used in plant biological control. American- Eurasian Journal of Sustainable Agriculture, 1(1), 7–12. Retrieved from http://www.aensiweb.
net/AENSIWEB/aejsa/aejsa/2007/7-12.pdf Hofstetter, V., Buyck, B., Eyssartier, G., Schnee, S., &
Gindro, K. (2019). The unbearable lightness of sequenced-based identification. Fungal Diversity, 96(1), 243-284. https://doi.org/10.1007/s13225- 019-00428-3
Islam, M., Al-Hashimi, A., Ayshasiddeka, M., Ali, H., El Enshasy, H. A., Dailin, D. J., … Yeasmin, T.
(2022). Prevalence of mycorrhizae in host plants and rhizosphere soil: A biodiversity aspect. PLoS ONE, 17(3), e0266403. https://doi.org/10.1371/
journal.pone.0266403
Jakobi, M., Winkelmann, G., Kaiser, D., Kempter, C., Jung, G., Berg, G., & Bahl, H. (1996). Maltophilin:
A new antifungal compound produced by Stenotrophomonas maltophilia R3089. Journal of Antibiotics, 49(11), 1101–1104. https://doi.
org/10.7164/antibiotics.49.1101
Jankiewicz, U., Brzezinska, M. S., & Saks, E. (2012).
Identification and characterization of a chitinase of Stenotrophomonas maltophilia, a bacterium that is antagonistic towards fungal phytopathogens. 113(1), 30–35. https://doi.
org/10.1016/j.jbiosc.2011.08.023
John, N., & Thangavel, M. (2017). Stenotrophomonas maltophilia: A novel plant growth promoter and biocontrol agent from marine environment.
International Journal of Advanced Research, 5(4), 207–214. https://doi.org/10.21474/
ijar01/3797
Kai, M., Effmert, U., Berg, G., & Piechulla, B. (2007).
Volatiles of bacterial antagonists inhibit mycelial growth of the plant pathogen Rhizoctonia solani.
Archives of Microbiology, 187(5), 351–360.
https://doi.org/10.1007/s00203-006-0199-0 Kalman, B., Abraham, D., Graph, S., Perl-Treves, R.,
Harel, Y. M., & Degani, O. (2020). Isolation and identification of Fusarium spp., the causal agents of onion (Allium cepa) basal rot in Northeastern Israel. Biology, 9(4), 69. https://doi.org/10.3390/
biology9040069
Koltai, H., & Kapulnik, Y. (2010). Arbuscular mycorrhizas:
Physiology and function. Dordrecht: Springer.
https://doi.org/10.1007/978-90-481-9489-6 Koske, R. E., & Gemma, J. N. (1989). A modified procedure
for staining roots to detect VA mycorrhizas.
Mycological Research, 92(4), 486–488. https://
doi.org/10.1016/S0953-7562(89)80195-9 Ladygina, N., Henry, F., Kant, M. R., Koller, R., Reidinger,
S., Rodriguez, A., … Wurst, S. (2010).
Additive and interactive effects of functionally dissimilar soil organisms on a grassland plant community. Soil Biology and Biochemistry, 42(12), 2266–2275. https://doi.org/10.1016/j.
soilbio.2010.08.027
Leslie, J. F., & Summerell, B. A. (2006). The Fusarium laboratory manual. Blackwell Publishing. https://
doi.org/10.1002/9780470278376
Mahdi, O., Eklund, B., & Fisher, N. (2014). Laboratory culture and maintenance of Stenotrophomonas maltophilia. Current Protocols in Microbiology, 32, 1–9. https://doi.org/10.1002/9780471729259.
mc06f01s32
Maiti, S. K., & Ghosh, D. (2020). Plant–soil interactions as a restoration tool. In Climate Change and Soil
Interactions (pp. 689-730). Elsevier. https://doi.
org/10.1016/b978-0-12-818032-7.00024-2 Marinho, F., Da Silva, I. R., Oehl, F., & Maia, L. C. (2018).
Checklist of arbuscular mycorrhizal fungi in tropical forests. Sydowia, 70, 107–127. https://
doi.org/10.12905/0380.sydowia70-2018-0107 Nakayama, T., Homma, Y., Hashidoko, Y., Mizutani,
J., & Tahara, S. (1999). Possible role of xanthobaccins produced by Stenotrophomonas sp. strain SB-K88 in suppression of sugar beet damping-off disease. Applied and Environmental Microbiology, 65(10), 4334–4339. https://doi.
org/10.1128/aem.65.10.4334-4339.1999 Nottingham, A. T., Turner, B. L., Winter, K., Chamberlain,
P. M., Stott, A., & Tanner, E. V. J. (2013). Root and arbuscular mycorrhizal mycelial interactions with soil microorganisms in lowland tropical forest. FEMS Microbiology Ecology, 85(1), 37–
50. https://doi.org/10.1111/1574-6941.12096 Ondov, B. D., Bergman, N. H., & Phillippy, A. M. (2011).
Interactive metagenomic visualization in a Web browser. BMC Bioinformatics, 12(1), 385. https://
doi.org/10.1186/1471-2105-12-385
Parihar, M., Rakshit, A., Rana, K., Tiwari, G., & Jatav, S.
S. (2020). The effect of arbuscular mycorrhizal fungi inoculation in mitigating salt stress of pea (Pisum sativum L.). Communications in Soil Science and Plant Analysis, 51(11), 1545–1559.
https://doi.org/10.1080/00103624.2020.178491 7
Pereira, C. M. R., da Silva, D. K. A., Goto, B. T., Rosendahl, S., & Maia, L. C. (2018). Management practices may lead to loss of arbuscular mycorrhizal fungal diversity in protected areas of the Brazilian Atlantic Forest. Fungal Ecology, 34, 50–58.
https://doi.org/10.1016/j.funeco.2018.05.001 Rajamohan, K., Udhayakumar, R., Sanjaygandhi, S.,
Kumar, L. V., Selvi, M. T., Sudhasha, S., &
Yuvarani, R. (2019). Efficacy of VAM fungi and
antagonistic bacteria against onion basal rot incited by Fusarium oxysporum f.sp. cepae.
Journal of Pharmacognosy and Phytochemistry, 8(2S), 413-418. Retrieved from https://
www.phytojournal.com/special-issue/2019.
v8.i2S.7409/efficacy-of-vam-fungi-and- antagonistic-bacteria-against-onion-basal-rot- incited-by-ltemgtfusarium-oxysporum- ltemgtfspltemgt-cepaeltemgt
Ranjbariyan, A. R., Shams-Ghahfarokhi, M., Kalantari, S., & Razzaghi-Abyaneh, M. (2011). Molecular identification of antagonistic bacteria from Tehran soils and evaluation of their inhibitory activities toward pathogenic fungi. Iranian Journal of Microbiology, 3(3), 140–146. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/
PMC3279816/
Sintayehu, A., Sakhuja, P. K., Fininsa, C., & Ahmed, S. (2011). Management of fusarium basal rot (Fusarium oxysporum f. sp. cepae) on shallot through fungicidal bulb treatment.
Crop Protection, 30(5), 560–565. https://doi.
org/10.1016/j.cropro.2010.12.027
Trabelsi, R., Sellami, H., Gharbi, Y., Krid, S., Cheffi, M., Kammoun, S., … Triki, M. A. (2017).
Morphological and molecular characterization of Fusarium spp. associated with olive trees dieback in Tunisia. 3 Biotech, 7(1), 28. https://
doi.org/10.1007/s13205-016-0587-3
van der Heijden, M. G. A., & Hartmann, M. (2016).
Networking in the plant microbiome. PLoS Biology, 14(2), e1002378. https://doi.
org/10.1371/journal.pbio.1002378
Zhou, J., Chai, X., Zhang, L., George, T. S., Wang, F., &
Feng, G. (2020). Different arbuscular mycorrhizal fungi cocolonizing on a single plant root system recruit distinct microbiomes. MSystems, 5(6), 00929-20. https://doi.org/10.1128/
msystems.00929-20
