Double action plant growth promotion microorganisms in suppressing Fusarium wilt disease and increase tomato production

Fusarium oxysporum f.sp lycopersici is a fungus that causes wilt disease in tomatoes, attacking nurseries to mature plants. This study aims to examine several plant growth-promoting microorganisms (PGPM) to control Fusarium wilt and increase tomato production. The study was designed with a completely randomized design in a screen house with four treatments and three experimental units. The treatments tested were mycorrhiza 5 g per plant (M), a mixed solution containing Bacillus sp., Pseudomonas sp., and Trichoderma sp. 10 ml per plant (PGPM1), and combination treatment M and PGPM1 (PGPM2). The study showed a more extended incubation period of the pathogen-causing diseased plants with the treatment of PGPM2 compared to other treatments. The lower intensity of wilt disease was also found in the PGPM2 treatment. Consistently the same treatment also had a better effect on the number of leaves, plant height, and fresh tomato fruit production than other treatments. This study showed a significant effect of PGPM in controlling Fusarium wilt and increasing tomato production.


Introduction
Tomato is an essential commodity for the domestic market in Indonesia.Tomatoes are widely used for food and beverage processing and even the cosmetic industry.The wide use of tomatoes makes high demand for this commodity.Tomato cultivation in Central Sulawesi is still constrained by wilt disease caused by Fusarium oxysporum f.sp lycopersici (FOL).The fungus is a soil-borne pathogen that causes wilt in tomatoes that has different physiological race worldwide [1][2][3][4].
One of the PGPMs is the Plant Growth Promoting Fungi (PGPF), fungus that can be used as a biological control agent.The two types of PGPF commonly used are mycorrhiza and Trichoderma sp.Arbuscular mycorrhiza is an obligate fungus antagonistic to plant pathogens [10,11].The symbiosis between plant roots and soil fungi has many benefits in agriculture, including increasing water absorption, nutrient status of plants, and plant resistance to diseases and other unfavourable conditions [12].Trichoderma sp., is an antagonistic fungus that can suppress the growth of soil-borne pathogenic 1253 (2023) 012024 IOP Publishing doi:10.1088/1755-1315/1253/1/012024 2 fungi.Several types of Trichoderma sp.produce siderophores that chelate iron and stop the growth of other fungi due to competition in taking up nutrients [13,14].Many studies have examined the effectiveness of mycorrhiza and Trichoderma sp., in suppressing the development of pathogens [15][16][17].
Another component of PGPM is plant growth-promoting rhizobacteria (PGPR).These bacteria are around plant roots and are involved in triggering plant growth and development directly and indirectly.Bacillus sp., and Pseudomonas sp. are commonly used [18].
The effectiveness of Bacillus sp. and Pseudomonas sp. have been the subject of numerous investigations in suppressing the development of pathogens, which have the potential to inhibit the pathogen of tomatoes [19][20][21].This study aims to analyze and compare the effect of addition plant growth-promoting microorganisms (PGPM) in suppressing Fusarium wilt and promote tomato growth and production.

Study site and experimental design
The study was carried out at the screen house and Laboratory of Phytopathology, Faculty of Agriculture, Tadulako University, Palu, Central Sulawesi, Indonesia.A completely randomized design (CRD) was used in this study, with four treatments, five replications, and three experimental units.The following were the experimental treatments: control (C, without treatment), Arbuscular mycorrhizal treatment 5 g pot -1 (M); Treatment with Bacillus sp., Pseudomonas sp., and Trichoderma sp., 10 ml pot -1 (PGPM1); treatment with Bacillus sp., Pseudomonas sp., Trichoderma sp., 10 ml pot -1 and mycorrhiza 5 g pot -1 (PGPM2).

Plant, FOL, and PGPM preparation
The research materials used were superior hybrid varieties of tomato seeds.Tomato seeds were sown in seedbeds containing sterile sandy soil (1:1 = v:v) for 14 days and showered with sufficient water.Seedling maintenance was carried out for up to two weeks and already has 3-4 leaves, then the tomatoes are transferred to the pot containing 10 kg of sterile soil according to the treatment.
FOL isolates were obtained from tomato showing symptoms of Fusarium wilt in Labuan village, Central Sulawesi, Indonesia.Isolation was carried out using Potato Dextrose Agar (PDA, HIMEDIA, USA).Colonies that emerged were then purified to become a single colony.Microscopic observations were made by making slide cultures to observe the fungal structure.The culture slides were observed under a microscope.Identification of fungi refers to the book The Identification of Fungi [22].

Pathogenicity test of FOL on tomatoes
Three weeks old tomato seedlings were inoculated with FOL suspension with a spore density of 10 6 ml - 1 .As much as 20 ml of FOL suspension was injected into the tomato root area in the pot.This test was carried out with five replications.Wilt symptoms in tomatoes were observed every day after inoculation until the plants died.

Incubation period and severity of fusarium wilt disease
The incubation period was observed every day since infection with the FOL on the plants until wilting symptoms appeared.The severity of the disease is known by observing the external symptoms of the plants.Observations were made every day after FOL inoculation.Disease intensity is calculated using the formula as follows: Where DS is the disease intensity, n is the number of plants observed, v is the disease intensity score, N is the highest score, and Z is the total number of plants.The disease severity score refers to Table 1.Leaves turn yellow and wilting (40% showed wilt symptoms) 3 Wilt, yellowing and browning (60% showed wilt symptoms) Plants wilt, yellow-brown discoloration more pronounced, and whole plant begins to die (80% showed wilt symptoms) 5 The whole plant dries up and dies with wilting (100% showed wilt symptoms)

Tomato growth and yield
Observation of tomato growth includes observation of plant height and the number of leaves.Observations began 3 until 11 weeks after planting.The growth and yield of tomatoes were measured by the number of leaves, plant height and fresh fruit weight.

Data analysis
Data on incubation period, disease severity, number of leaves, plant height, and fruit weight were analyzed by analysis of variance (ANOVA).If the treatment had a significant effect, it was continued with the least significant difference test (LSD) at the 5% level.Statistical analysis were done with R software [24] with the help of the R Studio interface application [25] using the Agricolae package [26].

Pathogenicity Test
Pathogenicity test results showed wilt disease symptoms began to appear six days after infection (Figure 1).The pathogenicity test showed wilting disease symptoms marked by the lower leaves turning yellow and stunted.F. oxysporum f.sp.lycopersici is the agent that causes Fusarium wilt disease in tomatoes, which attacks plants through the roots, and vascular tissue develops in the xylem, inhibits the movement of water and causes wilting, then causes the plants to die [27,28].

Incubation period of FOL
The control treatment showed the fastest incubation period compared to other treatments.The treatment of mycorrhiza, PGPM1 and PGPM2 had incubation periods of 19, 23 and 28 days, respectively (Table 2).

Table 2. The incubation period of FOL in tomatoes.
Note: Data are mean.The least significant difference test at level P<0.05 finds no statistically significant differences between numbers that are preceded by the same letter.
The time between being exposed to the pathogen and the beginning of disease symptoms is known as the incubation period.The disease's incubation period can be influenced by the level of plant susceptibility and the environment.Tomatoes infected with the pathogen showed symptoms of FOL with an incubation period of 12-28 days after infection.The fastest incubation period occurred in the control plants.The pathogen developed faster in plant tissues may be caused by plant susceptibility or due to the absence of antagonistic microbes that can protect plant roots from pathogens.Symbiotic roots with mycorrhizae broadly influence pathogenic microorganisms [11].Other studies described the application of Bacillus sp to potato tubers through the immersion method can delay the incubation period for seven days, and the treatment of Bacillus sp. and Pseudomonas sp. can prevent de wilting symptoms in potatoes for three days longer than controls [29].

Treatments
Incubation period (day) Control

Wilt disease severity
In the eleventh week, the disease severity increased by 88.22% in the control treatment, while in mycorrhiza, PGPM 1 and PGPM 2 treatments have limited disease by about 20.54%, 17.26%, and 13.44%, respectively.The average disease intensity in each treatment is presented in Table 3.
Table 3.The effect of treatment on the disease severity of tomatoes.
Note: Data are mean.The number in brackets is the transformation value of √x; The least significant difference test at level P<0.05 finds no statistically significant differences between numbers that are preceded by the same letter.
Table 3 shows that the control treatment significantly sever than other treatments.The PGPM2 treatment, which contained the mycorrhiza Trichoderma sp., Pseudomonas sp., and Bacillus sp., has the lowest effect on plant disease severity.Another study by Pentury [30] showed that mycorrhiza applied one week before Fusarium sp. have a good effect in increasing plant resistance.
Plant growth-promoting rhizobacteria (PGPR) can adapt to plants stressed by pathogen attacks.Mycorrhizae and PGPR can control vary of pathogens directly through competition and antibiosis and indirectly through chemical and physiological responses by changing enzyme activity and increasing chemical compounds, which can inhibit the development of pathogens [7,10,31].Mycorrhiza protects plant roots by infecting the plant root cortex and helping plants absorb nutrients needed by plants, thus increasing plant resistance to pathogen attack by releasing compounds that can prevent infection by pathogens such as phenols and phytoalexins [10,32,33].
Plant growth-promoting rhizobacteria (PGPR) has antifungal properties and interferes with the production of pathogenic toxins [31].Apart from acting as a biocontrol against soil pathogens, PGPR also produces substances that can control pathogens, such as siderophores and antibiotics, which can indirectly affect plant growth [7].Similarly, Trichoderma sp. has been studied widely as an antagonistic fungi against plant pathogens [14].

Tomato Growth and Yield
Plant growth and yield showed variation among the four treatments.The results showed that the treatments affected the growth and yield in tomatoes.Treatment PGPM2 has significantly higher fresh fruit tomato compared to other treatments (Table 4).Table 4.The effect of treatment on growth and yield of tomatoes.
Note: Data are mean.The number in brackets is the transformation value of √x; The least significant difference test at level P<0.05 finds no statistically significant differences between numbers that are preceded by the same letter.

Treatments
The disease severity, week after inoculated with FOL (%) The role of plant growth promotion rhizobacteria and rhizo-fungi that used in this study have been studied widely.Mycorrhizal roots have more significant energy metabolism, so that roots are active in absorbing P and N to stimulate plant growth which in turn helps absorb nutrients and water for photosynthetic needs [34].In addition, mycorrhiza also triggers an increase in the hormones cytokinin and auxin, which these two hormones play a role in cell differentiation and elongation [35].
Plant growth-promoting rhizobacteria (PGPR) can increase plant growth by regulating nutritional and hormonal balance, producing growth regulators and dissolving nutrients [36].The PGPR group of bacteria, such as Bacillus sp. and Pseudomonas sp., can dissolve phosphate and produce IAA hormones, increasing plant growth [37].Apart from acting as a biocontrol agent against soil pathogens, PGPR also produces substances that can control pathogens, such as siderophores and antibiotics and can indirectly affect plant growth [38].

Conclusion
Our study shows that plant growth-promoting microorganisms can suppress the development of pathogens and increase the production of tomatoes under a Fusarium wilt disease attack.The suppression of FOL is tremendous in applying PGPM in a complex combination of Bacillus sp., Pseudomonas sp., Trichoderma sp., and mycorrhiza (PGPM2) compared to other treatments.The PGPM2 also suppressed the development of FOL effectively than other treatments by delaying the disease incubation period of 16 days compared to the control and had lower disease severity.Simultaneously, tomato also has higher production in the treatment of PGPM2 compared to others.