Profiling of secondary metabolites produced by Pseudomonas sp. isolate PY-122 and PY-142 as biocontrol agent againts fusarium wilt disease on chili

Perfil de metabólitos secundários produzidos por Pseudomonas sp. isolado PY-122 e PY-142 como agente de biocontrole contra a doença de fusarium wilt em pimentas

Abstract

Fusarium wilt is an important disease on chili plants caused by the fungus Fusarium oxysporum. This study aims to determine the effectiveness of secondary metabolites from 2 Pseudomonad isolates in controlling Fusarium wilt in chili plants and their effect on chili plant growth. This research was conducted in two stages, namely in vitro, which was carried out in the plant health laboratory and greenhouse, Faculty of Agriculture, Universitas Pembangunan Nasional Veteran Jawa Timur, and in vivo which was carried out in chili plantations known to be endemic to Fusarium wilt in Menganti, Gresik. The research design used in this study was Completely Randomized Design for in vitro testing and Randomized Group Design for in vivo testing by testing 9 treatments repeated 3 times. The treatment tested was control, secondary metabolite application of Pseudomonas isolates PY-122 and PY-142 with concentrations of 20% 30%, 40% and 50%. The variables observed were inhibition of secondary metabolites on the growth of Fusarium fungus, disease incubation period, disease severity index, and plant growth. Sequencing results with 16rRNA gene markers for both Pseudomonas isolates showed similarities to Pseudomonas sp. The results showed that the treatment of secondary metabolite PY-142 with a concentration of 50% showed the highest consistent inhibition. In the greenhouse and field tests, the two isolates slowed down the disease incubation period and development compared to controls. In agronomical observations, the average plant height, number of leaves, root length, and appearance of the first flower on the treated plants were higher and more numerous than the control plants.

Keywords:
Fusarium wilt; Fusarium oxysporum ; chili; secondary metabolites; Pseudomonas sp

Resumo

A murcha de Fusarium é uma doença importante em plantas de pimenta causada pelo fungo Fusarium oxysporum. Este estudo tem como objetivo determinar a eficácia de metabólitos secundários de 2 isolados de Pseudomonas no controle da murcha de Fusarium em plantas de pimenta e seu efeito no crescimento da planta de pimenta. Esta pesquisa foi conduzida em duas etapas, a saber, in vitro, que foi realizada no laboratório de saúde vegetal e estufa, Faculdade de Agricultura, Universitas Pembangunan Nasional Veteran Jawa Timur, e in vivo, que foi realizada em plantações de pimenta conhecidas por serem endêmicas para a murcha de Fusarium em Menganti, Gresik. O delineamento de pesquisa usado neste estudo foi o Delineamento Completamente Randomizado para testes in vitro e o Delineamento de Grupo Randomizado para testes in vivo, testando 9 tratamentos repetidos 3 vezes. O tratamento testado foi controle, aplicação de metabólito secundário de isolados de Pseudomonas PY-122 e PY-142 com concentrações de 20%, 30%, 40% e 50%. As variáveis ​​observadas foram inibição de metabólitos secundários no crescimento do fungo Fusarium, período de incubação da doença, índice de severidade da doença e crescimento da planta. Os resultados do sequenciamento com marcadores do gene 16rRNA para ambos os isolados de Pseudomonas mostraram similaridades com Pseudomonas sp. Os resultados mostraram que o tratamento do metabólito secundário PY-142 com uma concentração de 50% apresentou a maior inibição consistente. Nos testes de estufa e campo, os dois isolados retardaram o período de incubação da doença e o desenvolvimento em comparação aos controles. Em observações agronômicas, a altura média das plantas, o número de folhas, o comprimento da raiz e o aparecimento da primeira flor nas plantas tratadas foram maiores e mais numerosos do que nas plantas de controlo.

Palavras-chave:
murcha de Fusarium; Fusarium oxysporum ; pimenta; metabólitos secundários; Pseudomonas sp

1. Introduction

Chili (Capsicum annum L.) is a herbaceous plant with a spicy fruit taste because it contains capsaicin. Chilies contain many nutrients and vitamins, including calories, protein, fat, carbohydrates, calcium, vitamins A, B1, and C (Chakrabarty et al., 2017). Indonesia chili production in 2024 will reach 1.334.219 tones, an increase of 23.97% compared to 2023 (Indonesian Ministry of Agriculture, 2020). One of the primary diseases that attack chili plants is a mild disease caused by the fungus Fusarium oxysporum, a soil-borne fungus. This pathogen causes damping off of seedlings (serious seed germination) and causes abnormal growth. This pathogen infects young roots, stem vessels, inhibiting air and nutrient transport (Miller et al., 2008).

Secondary metabolites are organic compounds that are indirectly involved in the growth, development, and reproduction of organisms and are usually formed during the end or near the stationary stage of growth of organisms (Kliebenstein and Osbourn, 2012; Li et al., 2016) secondary metabolite compounds produced by beneficial microbes can be used to control plant-disturbing organisms (Keswani et al., 2019). One of the beneficial microbes that produce secondary metabolites is Pseudomonads fluorescens. The Pseudomonads fluorescens group of bacteria can form secondary metabolites, namely siderophores which can chelate Fe ions (Singh et al., 2022). Pseudomonas bacterial strain as a biocontrol agent can produce several secondary metabolites, including hydrogen cyanide (HCN); 2,4-diacetylphloroglucinol (DAPG); phenazines; pyrrolnitrin; pyolutheorin; 2-hexyl-5-propyl-alkylresorcinol; cyclic) lipopeptides (Höfte, 2021).

The Pseudomonas sp. bacteria group that can be used as a biocontrol agent, especially for soil-borne pathogens can produce antibiotic compounds, such as antifungal, antibacterial, antitumor, and antiviral (Haggag and Abo El Soud, 2012). Disease suppression mechanism by Pseudomonas sp., namely competition in taking up nutrients and space (Kamilova et al., 2005), antibiosis (Haas and Défago, 2005; Raaijmakers et al., 2002), and induce systemic resistance (Bakker et al., 2007). The bacteria P. fluorescens WCS365 can suppress the growth of microconidia of the soil-borne fungus Fusarium oxysporum f.sp.radicis-licopersici on tomatoes (Kamilova et al., 2008). In addition, this bacterial isolate can colonize the roots of potatoes, tomatoes, radishes, and wheat (Brand et al., 1991; Dekkers et al., 1998)—however, the utilization of secondary metabolites from Pseudomonas sp. Only a little has been done to control F. oxysporum which causes wilt disease in chili peppers, so it is necessary to study the application of secondary metabolites of Pseudomonas sp. against suppression of wilt disease in chilies. This study aims to examine the effectiveness of secondary metabolites from 2 isolates of Pseudomonas sp. against wilt disease and its effect on the growth of chili plants.

2. Material and Methods

2.1. Fungi pathogens used

Fusarium oxysporum f. sp. capsici strain YW201 was obtained from the Plant Health Laboratory collection and was molecularly identified as DSMZ 19286 by DNA barcoding. Pathogenic ability of F. oxysporum f. sp. capsici strain YW201 was tested by referring to Hami et al. (2021). The modified method involved soaking cayenne pepper plants, which had their roots injured, in 10 mL suspension of F. oxysporum f. sp. capsici strain YW201. Subsequently, these plants were planted in soil that had also been treated with a suspension of F. oxysporum f. sp. capsici strain YW201. After five days of incubation, the chili plants exhibited symptoms such as wilting, inward rolling of leaves, chlorosis, and stunted growth. F. oxysporum f. sp. capsici strain YW201 was cultured and stored on eight different types of vegetable extract media, including V8 Juice Agar Media

2.2. Isolation and selection of Pseudomonas genus bacteria with biocontrol activity

Samples of healthy chili plant roots among diseased chili plants from Wajak Village, East Java Province, Indonesia were collected. The soil attached to the roots was collected for the collection of bacterial isolates of the genus Pseudomonas, part of the rhizosphere and rhizoplane using the stratified dilution method referring to Somasegaran and Hoben (1994) and physiological identification referring to the method of Schaad et al. (2001). The purified Pseudomonas isolate was stored in LB oblique agar at 4°C and 20% glycerol -80°C.

Inhibition test of Pseudomonas isolates obtained against F. oxysporum f. sp. capsici strain YW201 was carried out using the dual culture method referring to Xiong et al., (2004) on Potato Dextrose Agar media. On the 3rd day of incubation, the inhibition of the bacterial isolates against F. oxysporum f. sp. capsici strain YW201 using Formula 1 referring to Xiong et al. (2004):

$I\left(\%\right)=\frac{DC-DT}{DC}\times 100\%$ (1)

Keterangan:

I = Inhibition percentage (%)

DC = Diameter of fungal growth in the control treatment

DT = Diameter of fungal growth in treatment

2.3. Species identification of Pseudomonas by 16S rRNA gene sequencing and phylogenetic analysis

Isolation of genomic DNA of Pseudomonas bacteria was carried out following standard procedures of the Presto™ Mini gDNA Bacterial Kit (Geneaid, USA). 16S rRNA gene amplification was performed using primers 63F and 1387R (Marchesi et al., 1998). The final concentration of the PCR reaction consisted of 1 L of GoTaq® Green Ready Mix (Promega, USA), 0.5M of each primer, 10 µg of genomic DNA, and nuclease-free water to a volume of 50 µL. PCR amplified product sequenced commercially by Angler Biochemlab, Indonesia. The bacterial lineage sequences available in NCBI were used to align and compare sequence data using BLAST. Accession numbers are allocated after submitting the sequence to the NCBI GenBank database (Yasmin et al., 2016).

2.4. Mass spectrometric analysis of secondary metabolite

Detection and identification of secondary metabolites of the best-selected Pseudomonas isolates was carried out with the help of LCMS/MS. The Pseudomonas isolate suspension obtained was shaken at 30 ± 2°C for 24 and 48 hours. The bacterial cell pellet was then removed by centrifugation at 4°C for 15 minutes at 10,000 rpm. The pH of the supernatant was reduced to 3.0 using 3M HCl solution before extraction with 500 mL ethyl acetate. The extraction step was repeated with 300 mL ethyl acetate. The residue was dissolved in 5 mL of LCMS-grade methanol and analyzed for LCMS/MS using a mass spectrometer (LTQ XL Linear Ion Trap Mass Spectrophotometer, Thermo Scientific, USA). Samples were filter-sterilized and injected via direct syringe pump at a flow rate of 8 μL min ̄1. Samples were scanned in positive and negative ion full scanning mode (m/z mass scan range 50-2000) with source and capillary voltages of 4.8 kV and 23 V respectively with the capillary temperature of 350°C and 30 arbitrary units in both modes scan.

2.5. Inhibition of Pseudomonas sp. secondary metabolites against F. oxysporum f. sp. capsici in vitro

The best selected Pseudomonas isolate suspension was shaken at 150 rpm at room temperature for 72 hours. The bacterial cell pellet was then removed by centrifugation at 4°C for 15 minutes at 10,000 rpm to obtain secondary metabolites. The supernatant was then filtered on a 0.22 µm syringe filter (Elita et al., 2013). The supernatant and V8 juice media were mixed using a pour plate with a ratio of 9:1 and then compacted in a petridish. The supernatant was mixed in several concentration treatments, namely control (without secondary metabolite suspension) 200 mL/L, 300 mL/L, 400 mL/L, and 500 mL/L. Inoculum F. oxysporum f. sp. capsici with a diameter of 6 mm were placed on the surface of the solid media and incubated at room temperature (Khalil et al., 2022)

Inhibition was measured qualitatively by observing the hyphae abnormality of F. oxysporum f. sp. capsici, and quantitatively by calculating the diameter and spore density of F. oxysporum f. sp. capsici. Diameter F. oxysporum f. sp. capsici was measured by Formula 2 referring to (Seema and Devaki, 2012) and spore density was measured using a haemocytometer with Formula 3 referring to Gabriel and Riyanto (1989):

$P\left(\%\right)=\frac{DK-DP}{DK}\times 100\%$ (2)

Note:

P (%) = Percentage of growth inhibition of pathogenic fungi

DK = Diameter of fungal growth in the control treatment

DP = Diameter of fungal growth in treatment

$C=\frac{t}{\left(nx\mathrm{0,25}\right)}\times {10}^6$ (3)

Note:

C = Spore density per mL of solution

t = The total number of spores in the sample box has been observed

n = Number of sample boxes: 5 large boxes x 16 small boxes

0,25 = correction factor for using a small-scale sample box on a hemocytometer

2.6. Disease assessment of Fusarium wilt suppressed by Pseudomonas secondary metabolites

Fusarium wilt disease was analyzed by conducting experiments in greenhouses and in the field. The greenhouse experiment was carried out at the Green House of the Faculty of Agriculture, Universitas Pembangunan Nasional Veteran Jawa Timur, and the field experiment was carried out in the Menganti area, East Java Province, Indonesia on fields endemic to Fusarium wilt of chili plants. A total of 10 mL of suspension of F. oxysporum f. sp. capsici poured on the soil planting medium and then incubated for 14 days in polybags. Chili seedlings aged 4 week after planting (WAP) were then planted in soil media that had been inoculated with F. oxysporum f. sp. capsici. The best Pseudomonas isolate secondary metabolite suspension was prepared for the control treatment (without secondary metabolite suspension) 200 mL/L, 300 mL/L, 400 mL/L, and 500 mL/L as much as 10 mL per treatment unit. The treatment was repeated 7 times.

Analysis of Fusarium wilt includes the incubation period Campbell and Madden (1990), the percentage of disease incidence (Bowen, 2007), the level of infection or virulence of the pathogen (Van der Plank, 1963). The incubation period was calculated from the time of the first disease inoculation until the first symptoms appeared, the disease severity index was calculated using the Formula 4 and 5:

$DII\left(\%\right)=\frac{(\Sigma \left(\text{ni}\times \text{vi}\right)}{\left(\text{Z}\times \text{N}\right)}\times 100\%$ (4)

Note:

DII = Disease Intensity Index(%)

ni = number of affected plant parts in category i

vi = numeric value in each category

N = number of plants/part of sample plant

Z = highest attack category value

$r=\frac{\mathrm{2,}30259}{\text{t}2-\text{t}1}(\log 10\frac{x2\left(1-x1\right)}{x1\left(1-x2\right)})$ (5)

Note:

r = Infection rate

t2 = 2^nd observation time

t1 = 1^st observation time

X2 = proportion of diseased leaves after t2

X1 = proportion of diseased leaves at the initial interval

2.7. Statistical analysis

Data were analyzed by analysis of variance at 5% significance level. Significant differences between treatments were further tested using Duncan's Multiple Range Test (DMRT) at the same significance level.

3. Result and Discussion

3.1. Isolation and selection of Pseudomonas genus bacteria with biocontrol activity

A total of 130 bacterial isolates were obtained from the rhizosphere and rhizoplane. The 130 bacterial isolates were then identified morphologically and physiologically following the method of Schaad et al. (2001) to obtain bacteria of the genus Pseudomonas. Identification of the bacterial genus based on morphology and physiology is presented in Table 1 and Table 2.

Table 1
Morphological characteristics of bacteria isolated from rhizosphere and rhizoplane.

Colony				Number of Isolates
Shape	Color	Surface	Edge
Rounded	Yellowish	Convex	Undulate	43
Rounded	White	Convex	Entire	32
Rounded	Pink	Flat	Entire	5
Oval	Yellowish	Convex	Undulate	21
Oval	White	Convex	Entire	20
Irregular	White	Convex	Entire	9

Table 2
Physiological characteristics of bacteria isolated from rhizosphere and rhizoplane.

Genus	Gram	OF	YDC	KB	Ed	Number of Isolates
Corynebacterium	+	F			-	5
Pseudomonas	-	O	+	+	-	12
Bacillus	+	O		+	+	27
Xanthomonas	-	O	+	-	-	25
Erwinia	-	F	-		-	23
Unknown	+	-	-	-	-	28

• Note: OF = Oxidative/Fermentative Test; YDC = Yeast Dextrose CaCO₃ Test; KB = King’s B Media Test; Ed = Endospore Test.

The bacteria isolate is qualitatively identified by morphological and physiological characteristics using Schaad et al. (2001) method to collect Pseudomonas genus bacteria. Table 1 shows different morphological characteristics of bacteria isolates. The dominant morphological characteristics of isolates are rounded shape, white color, convex surface, and entire edge. In the physiological test, 12 isolates were purified as Pseudomonas genus bacteria. The Pseudomonas isolates had gram-positive cells, oxidatively metabolic, positive or had yellowish colonies in the YDC media cultured, and positive or had fluorescent pigment colonies cultured in King’s B media (Table 2).

3.2. Selection of Pseudomonas genus bacteria with biocontrol activity

12 bacterial isolates of the genus Pseudomonas were obtained from morphological and physiological characterization. Bacterial isolates of the genus Pseudomonas were then selected to determine their antagonistic ability against the pathogen F. oxysporum f. sp. capsici. Inhibition of the pathogen F. oxysporum f. sp. capsici by bacterial isolates of the genus Pseudomonas are presented in Table 3.

Table 3
Inhibition of F. oxysporum f. sp. capsici by Pseudomonas Isolates.

Treatments	Diameter (cm)	Percentage of Inhibition (%)
Control without I-PY	6.30	0.00	-
I-PY079	4.87	22.70	ab
I-PY085	5.32	15.56	ab
I-PY088	5.70	9.52	a
I-PY090	3.50	44.44	bc
I-PY098	2.90	53.97	bc
I-PY113	5.89	7.94	a
I-PY120	3.11	50.63	bc
I-PY121	3.56	43.49	b
I-PY122	2.17	65.56	c
I-PY128	4.44	29.52	ab
I-PY142	1.89	70.00	c
I-PY147	4.90	22.22	ab

• Note: I-PY = Pseudomonas Isolates.

Antagonism ability of the Pseudomonas isolates against F. oxyporum f. sp. capsici were selected using dual culture method. Based on the experiment (Table 3) the highest two number of F. oxysporum f. sp. inhibition was in the treatment of isolate code I-PY122 for 65,56% and isolate code I-PY142 for 70,00%. Isolates I-PY122 and I-PY142 then were taken to be characterized using bacterial genomic DNA isolation in the Polymerase Chain Reaction method based on 16S rRNA gene amplification.

3.3. Molecular identification of Pseudomonas genus bacteria with biocontrol activity

Pseudomonas genus isolates with inhibition percentage of Fusarium oxysporum f. sp. capsici strain YW201. The highest isolates namely I-PY122 and I-PY142 were identified molecularly. DNA amplification was carried out using 16S rRNA primers, then separated by agarose gel electrophoresis and visualized under UV light. Visualization of agarose gel electrophoresis PCR results is presented in Figure 1. BLAST analysis of the 16S rRNA genes showed that isolates I-PY122 and I-PY142 have 100% similarity with Pseudomonas sp. FBF114 and Pseudomonas p. FBF57 (Figure 1, Table 4).

Figure 1
Visualization of agarose gel electrophoresis amplified the 16S rRNA gene of I-PY122 and I-PY142 isolates in DNA Ladder 10.000 bp and a phylogenetic tree with 1000x repetition using Neighbor-Joining Method and Jones-Taylor-Thorthon (JTT) substitution model.

Table 4
Identification of Pseudomonas bacteria strain.

Bacterial Strain	Homology (%)	Species Database on GeneBank	Accession Number
I-PY122	100	Pseudomonas sp. FBF141	HG805785
I-PY142	100	Pseudomonas sp. FBF57	HG805731

3.4. Inhibition of Fusarium wilt by Pseudomonas secondary metabolites

The supernatant of 2 isolates of Pseudomonas sp. has been confirmed on molecular identification extracted for inhibition tests against the pathogen F. oxysporum f. sp. capsici. Inhibition of F. oxysporum f. sp. capsici by supernatant Pseudomonas sp. is presented in Table 5.

Table 5
Inhibition of F. oxysporum f. sp. capsici by Pseudomonas sp. Supernatant.

Treatments	Diameter (cm)	Percentage of Inhibition (%)
Control without S-PY	5.60	0.00	-
S-PY122 200 mL/L	5.30	5.36	a
S-PY122 300 mL/L	5.15	8.04	a
S-PY122 400 mL/L	5.12	8.57	a
S-PY122 500 mL/L	4.60	17.86	ab
S-PY142 200 mL/L	4.30	23.21	ab
S-PY142 300 mL/L	3.90	30.36	ab
S-PY142 400 mL/L	3.61	35.54	ab
S-PY142 500 mL/L	2.76	50.71	b

• Note: S-PY122 = Supernatant Pseudomonas sp. strain 122; S-PY142 = Supernatant Pseudomonas sp. strain 142.

Eight supernatants in different concentrations from isolate PY122 and PY142 were evaluated in the inhibition assays. All of the supernatant concentrations were able to inhibit F. oxyposrum f. sp. capsici proven by the inhibition percentages of F. oxysporum f. sp. capsici diameters. Table 5 shows 500 mL/L concentration of isolate PY142 supernatant was able to inhibit F. oxysporum f. sp. capsici growth until 50,71%. It is the highest number of inhibition percentages from all treatments and among the concentration of supernatant PY142. Furthermore, 500 mL/L of isolate PY122 supernatant was the highest percentage of inhibition among the concentration of supernatant PY122 which is 17,86%.

3.5. Growth and hyphae abnormality of F. oxysporum f. sp. capsici

Hyphae shape and spore density of F. oxysporum f. sp. capsici observed after treatment with several supernatant concentrations of Pseudomonas sp. Hyphae were observed at 400 times the magnification of the microscope. Spores were counted with a hemocytometer. There are abnormalities in hyphae and differences in the number of spores of F. oxysporum f. sp. capsici after being given treatment. Hyphal abnormalities of F. oxysporum f. sp. capsici is shown in Figure 2, and the spore densities are presented in Table 6.

Figure 2
Abnormality of F. oxysporum f. sp. capsici after treated by Pseudomonas sp. secondary metabolites: a) hyphae bend b) hyphae curl c) hyphae lysis d) hyphae shrink.

Table 6
Density of F. oxysporum f. sp. capsici (CFU/mL).

Treatment Code	Density of F. oxysporum f. sp. capsici (CFU/mL)		Density of F. oxysporum f. sp. capsici (LOG10)
Control without S-PY	3.12 x 108	d	8.49
S-PY122 200 mL/L	2.98 x 108	d	8.47
S-PY122 300 mL/L	2.87 x 107	c	7.46
S-PY122 400 mL/L	2.88 x 107	c	7.46
S-PY122 500 mL/L	2.77 x 107	c	7.44
S-PY142 200 mL/L	1.20 x 107	c	7.08
S-PY142 300 mL/L	8.92 x 106	b	6.95
S-PY142 400 mL/L	7.73 x 105	a	5.89
S-PY142 500 mL/L	5.82 x 105	a	5.76

• Note: S-PY122 = Supernatant Pseudomonas sp. strain 122; S-PY142 = Supernatant Pseudomonas sp. strain 142.

Table 5 and Figure 3 show the treatment of the supernatant concentration of Pseudomonas sp. able to prevent the colonization of the fungus Fusarium oxysporum f. sp. capsici strain YW201 In Figure 2 the hyphae of F. oxysporum f. sp. capsici experience fracture, curl, and lysis. Supported by the data in Table 6, the formation of spores of F. oxysporum f. sp. capsici was inhibited by the activity of Pseudomonas sp. isolates supernatant. The lowest spore density was at the treatment concentration of 400 mL/L and 500 mL/L supernatant of Pseudomonas sp. PY-142 with Log10 results of spore density of 5.76 and 5.89. While the highest spore density was in the control treatment and the treatment with a concentration of 200 mL/L supernatant of Pseudomonas sp. PY-122 with Log10 results of spore densities of 8.49 and 8.47.

Figure 3
Inhibition of F. oxysporum f. sp. capsici after S-PY122 and S-PY142 treatments.

3.6. Mass spectrometric analysis of Pseudomonas sp. secondary metabolites

Chromatographic analysis was carried out using LC-MS/MS at a voltage of 23 V. There were several molecular masses obtained at the peaks of the chromatogram, namely 157, 169, 183, 195, 197, 207, 211, 219, 233, 235, 245, 258, 267, 272, 283, 291, 301, 317, 319 and 329 m/z. The chromatogram results are presented in Figure 4.

Figure 4
Chromatogram of Pseudomonas sp. supernatant in 23 V.

3.7. Field analysis of Fusarium disease inoculated with Pseudomonas sp. supernatant

Field experiments were carried out to prove the secondary metabolites of Pseudomonas sp. can suppress the pathogen F. oxysporum f. sp. capsici. This experiment was carried out in green houses and Fusarium wilt endemic fields. The analysis includes Incubation Periods (IP), Disease Intensity Index (DII), and Infection Rate (r). Analysis of Fusarium wilt in green houses and Fusarium endemic fields is presented in Table 7 and Table 8.

Table 7
Fusarium wilt analysis at greenhouse experiment.

Treatment Code	Disease Assessment (30 days)
	IP (days)	Σ DII (%)	r Value
Control without S-PY	8	36.31	0.6743
S-PY122 200 mL/L	11	17.04	-0.3765
S-PY122 300 mL/L	11	15.32	-0.3698
S-PY122 400 mL/L	12	12.30	-0.3704
S-PY122 500 mL/L	12	12.30	-0.3865
S-PY142 200 mL/L	13	11.11	-0.5472
S-PY142 300 mL/L	13	3.3	-0.7796
S-PY142 400 mL/L	13	3.53	-0.7965
S-PY142 500 mL/L	17	3.53	-0.8739

• Note: S-PY122 = Supernatant Pseudomonas sp. strain 122; S-PY142 = Supernatant Pseudomonas sp. strain 142; IP = Incubation Period; DII = Disease Intensity Index.

Table 8
Fusarium wilt analysis at field experiment.

Treatment Code	Disease Assessment (30 days)
	IP (days)	DII (%)	r Value
Control without S-PY	0	86.32	0.9742
S-PY122 200 mL/L	10	57.04	-0.6763
S-PY122 300 mL/L	15	55.36	-0.6695
S-PY122 400 mL/L	15	52.36	-0.6705
S-PY122 500 mL/L	16	52.35	-0.6865
S-PY142 200 mL/L	20	31.15	-0.6473
S-PY142 300 mL/L	24	11.58	-0.9793
S-PY142 400 mL/L	25	10.42	-0.9862
S-PY142 500 mL/L	27	10.42	-0.9932

• Note: S-PY122 = Supernatant Pseudomonas sp. strain 122; S-PY142 = Supernatant Pseudomonas sp. strain 142; IP = Incubation Period; DII = Disease Intensity Index.

​4. Discussion

This experiment is aimed to profile Pseudomonas sp. secondary metabolites and screen the ability of Pseudomonas sp. secondary metabolites against wilt disease pathogen F. oxysporum f. sp. capsici in chili. Exploration of biocontrol potential bacteria was conducted in Wajak District, East Java Province, Indonesia. 130 bacteria isolates were collected from the rhizosphere and rhizoplane healthy chili plant. The bacteria isolate is qualitatively identified by morphological and physiological characteristics using Schaad et al. (2001) method to collect Pseudomonas genus bacteria. Table 1 shows different morphological characteristics of bacteria isolates. The dominant morphological character of isolates are rounded shape, white color, convex surface, and entire edge. In the physiological test, 12 isolates were purified as Pseudomonas genus bacteria. The Pseudomonas isolates had gram-positive cells, oxidatively metabolic, positive or had yellowish colonies in the YDC media cultured, and positive or had fluorescent pigment colonies cultured in King’s B media (Table 2).

The antagonism ability of the Pseudomonas isolates against F. oxyporum f. sp. capsici was selected using the dual culture method. The highest two numbers of F. oxysporum f. sp. inhibition were in the treatment of isolate code I-PY122 for 65,56% and isolate code I-PY142 for 70,00% (Table 3).

(Haggag and Abo El Soud, 2012) the bacterial isolate Pseudomonas sp. can produce antifungal compounds that can interfere with the growth of pathogenic fungi. In addition, these metabolites also produce other antibiotic compounds, namely, antibacterial, antitumor, and antiviral. The antifungal compound produced by the bacterium Pseudomonas sp., namely phenazines, pyoluteorin, oomycin A, sulphonamide, and pyocyanin. It also produces the antibiotic compound 2,4-diacetylfluoroglinol or DAPG, which plays a role in inhibiting the growth of fungi (Vacheron et al., 2013). Siderophores produced by the Pseudomonas fluorescens group of bacteria are also known to be able to inhibit several types of phytopathogenic fungi such as Phytophthora parasitica, Phythium ultimum, Fusarium oxysporum veri dianthi and Sclerotinia sclerotiorum (Páez et al., 2005).

Based on Table 2, the incubation period for all treatments slowed down the appearance of symptoms on chili plants compared to control plants without treatment. This was because the control plants did not apply secondary metabolites, so the plants did not have any defense against Fusarium fungal infection. The incubation period for the appearance of wilting symptoms in the chili seedbed without treatment is 6 days after inoculation (Soesanto, 2013). Applying secondary metabolite M1K2 with a concentration of 30% in soil media took the longest time to delay the appearance of wilting symptoms in greenhouses. The incubation period of Fusarium pathogens in the field with the treatment of secondary metabolites from bacteria PY122 with a concentration of 20% and 50% and PY142 with a concentration of 40% was longer than the control. Even treatment with PY122 at concentrations of 30 and 40% until the end of the observation did not cause symptoms. This is presumably because the secondary metabolites of the bacterial isolates can inhibit the growth of the Fusarium fungus due to the toxic compounds they produce. Thus extending the incubation period of disease emergence in plants. In addition, changing environmental conditions can affect disease development (Pokhrel, 2021).

Based on observations in all treatments, the disease progressed from day 5 to day 30, showing lower development than the controls. This shows that the administration of secondary metabolites can inhibit the development of wilt disease in the field. Treatment of secondary metabolite PY-122 with concentrations of 20, 30, and 40% from the beginning to the end of the observation showed no signs of wilting. This shows that the secondary metabolite PY 122 with concentrations of 20, 30, and 40% is the best in suppressing wilt in the field. Factors that affect the severity of plant diseases in the field are too dense spacing (O’Hara et al., 2016) and various environmental factors (Velásquez et al., 2018).

Acknowledgements

We gratefully acknowledge the generous research funding provided by the Directorate General of Higher Education, Research and Technology, Ministry of Education, Culture, Research and Technology

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Publication Dates

• Publication in this collection
  14 Oct 2024
• Date of issue
  2024

History

• Received
  20 Sept 2023
• Accepted
  01 Aug 2024