Arbuscular Vascular Mycorrhizes (MVA) to Control Wilt Disease in Tomato (Solanum lycopersicum L.)

Tomatoes continue to develop into an essential commodity in international trade. Suboptimal tomato production can be caused by pest and disease attacks (Ma et al., 2023). (Gatahi, 2020)  said, it was conveyed that tomato cultivation cannot be separated from various obstacles affecting its production. The pathogens that cause wilt disease in tomato plants are Fusarium oxysporum f. sp. lycopersici (Fol) and Ralstonia solanacearum (RS). FoL attacks can reach 60 to 70% (Srinivas et al., 2019) and even up to 85% (Sukorini et al., 2023). In comparison, RS attacks on tomato plants are 77 to 90% (Wamani et al., 2023). The genus Fusarium fungi includes several species of plant pathogens, among the most damaging phytopathogens worldwide, causing diseases in many crops, such as cereals and other crops (Todorović et al., 2023). It can harm over 100 essential cash crops such as banana, tomato, cucumber, M. charantia, watermelon, muskmelon, cotton and beans (Yan et al., 2023). In addition to causing diseases, these fungi produce a wide range of mycotoxins, which may be present in feed and food products. While Ralstonia solanacearum can infect a wide range of hosts, including banana, tobacco, potato, tomato, ginger, eggplant and chili (Ahmed et al., 2022). This bacterium can attack more than 200 plant species from 50 different families and cause losses of up to 100% (Garćia et al., 2019).

One alternative that can be used to control wilt disease in tomato plants is to use biological agents. The use of biological agents that live around the roots is greatly influenced by environmental conditions, especially soil humidity and temperature, so it is less effective. Germination of Fusarium proliferatum spores, a seed-borne pathogen of plants (Soni et al., 2021), showed that in vitro treatment with chemical pesticides was better than biological treatment using Pseudomonas fluorescens and Trichoderma harzianum. However, research (Murali Sankar et al.,  2019) shows that Pseudomonas fluorescens has a high population of 8.2x10⁵ cfu/g soil. Pseudomonas chlororaphis has better growth-promoting and disease-controlling properties in chickpeas with superior root colonization capabilities. Biological agents that have the potential to control wilt disease in tomato plants are mycorrhiza (Meddad-Hamza et al., 2023). On the other hand, the impact of mycorrhizal fungal colonization on legume plants can reduce wilt disease, showing changes in soil biological fertility, especially in terms of phosphate and nitrogen content, so that it can be used as a biofertilizer (Benelhadj Djelloul et al.,  2024). Applying mycorrhizal fungi to tomato plants can significantly affect the intensity of FoL attacks, reducing the intensity of attacks by 28.26%, starting from the initial disease severity of 69.85% (Meddad-Hamza et al., 2023). While controlling R. solanacearum, G. Mosseae could reduce disease severity from 55% at 20 days to 0%, but could not significantly reduce disease severity in other types. Research on the time of application and the number of spores given to reduce FoL or RS attacks has never been conducted. Related research on the existing application time is on oil palm plants (Hendarjanti and Sukorini, 2022). This research aimed to determine the interaction between the number of spores and the application time of Arbuscular Vascular Mycorrhiza (MVA) on the growth and control of wilting in tomato plants (Solanum lycopersici L.).

Place and Time
 
This research was conducted from August to November 2023 (rainy season) at the Agronomy Laboratory, the University of Muhammadiyah Malang and the Ngadaprejo Farmers’ Land, Junrejo District, Batu City, East Java. The altitude was 739 meters above sea level and the soil type was Andosol. The average temperature was 18- 23°C and the Relative humidity was 76-97%. The land for the research location was a former tomato plantation owned by a farmer, 87% of which was infected with wilt disease.
 
Research design
 
The research design was a randomized block design (RBD) arranged factorially. This study used two factors and the first was the dose of mycorrhizal administration (M) with three treatments consisting of M1 = mycorrhizal 25 spores/plant, M2 = mycorrhizal 50 spores/plant and M3 = mycorrhizal 75 spores/plant. The second factor was Mycorrhiza (T) application time, using two treatments consisting of T1 = when the tomato seeds were sown and T2 = when transplanting. So, there are six treatment combinations. Each treatment contained five plant samples. All treatment combinations were repeated 4 times. The varieties used are varieties that farmers usually plant, namely the F1-resistant variety, which was an introduced hybrid plant. The type of plant was a determinate plant.
 
Application of vesicular arbuscular mycorrhiza in tomato plants
 
The soil used for the planting medium in this study originated from the research area previously used for tomato cultivation, so it has the potential for pathogens to settle in the soil. The soil is then put in a polybag, sprayed with water and left for one week before planting. Then, Mycorrhiza of  25, 55 and 75 spores/ plants can be applied to the soil for each sample. The mycorrhiza used was a personal collection of mycorrhiza propagated in corn plants. Furthermore, the spores in the soil were counted using the method (Sasvári et al.,  2012) to determine the spore content in 100 grams of soil. Based on the number of spores contained in the soil, the application of 25/50/75 spores was determined.
 
Data analysis
 
Data were analyzed using ANOVA. If the treatment has a natural effect, it is tested further with the Duncan multiple range test at a 5% level.

Plant height (cm)
 
The treatment of giving the amount of mycorrhiza had a very significant effect on all plant ages. The results of further tests with DMRT at the 5% level are presented in Table 1.



Table 1 shows that treating 75 mycorrhizal spores at the sowing time achieved the longest plant height at all observation ages.
 
The number of leaves (Leaf blade)
 
The number of leaves shows a very significant interaction effect. The highest number of leaves at all observation ages in the treatment of mycorrhizal administration time was 75 spores during seedlings. This was not significantly different from the treatment of 75 spores during transplanting. The results of further tests with the DMRT test at the 5% level are presented in (Table 2).


 
Fruit weight (grams)
 
The fruit weight per plant was observed at tomato harvest 63 Days After Planting (DAP). Mycorrhizal and application time interacted with the fruit weight variable. The results of further tests with the DMRT test at the 5% level are presented in (Table 4).

Table 3 shows that 75 mycorrhizal spores at the time of transplanting produced the highest tomato fruit weight and were not significantly different from the treatment of giving 50 mycorrhizal spores at the time of sowing.




 
Root infection by mycorrhizae and disease incidence(%)
 
There was a significant interaction between the number of mycorrhizae applied and the time of mycorrhizae application, combined with root infection and disease incidence. The results of further testing with the DMRT test at the 5% level are presented in Table 4. Table 4 showed that the observation of the percentage of root infection of the treatment of giving 75 spores of mycorrhiza at the time of seeding resulted in the highest mycorrhiza root infection and was not significantly different from the treatment of applying 75 spores of mycorrhiza at the time of transplanting. At the age of 6 MST and 7 MST, the highest attack was in the treatment of 25 spores, which was given at the time of transplanting.

Mycorrhiza takes time to infect the roots of plants. Where mycorrhizal hyphae colonize the roots of tomato plants by forming external hyphae to expand the absorption area of tomato plant roots in obtaining water, nutrients and other nutrients, the infection process begins with germination in the soil and begins to form hyphae; the growing hyphae will penetrate the roots, which will later develop in the cortex. In roots that have been infected by mycorrhiza, arbuscules will form and from these arbuscules, mycorrhiza can help absorb nutrients needed by plants (Mohammadi et al., 2020). The mycorrhiza that is given has infected the roots of tomato plants and is actively carrying out mutualistic symbiosis with tomato plants; this can support the growth of tomato plants, especially plant height, so that there is a difference in tomato plant height between treatments. Other studies also explain that giving AMF Glomus sp. as much as 20 grams can increase the growth of castor oil plant seedlings, especially plant height. AMF with phosphate enzyme content can free P and N elements that were initially unavailable and then become available in the soil  (Zhang et al., 2014; Ettlili et al.,  2022). The nutrient N is a stimulant for plant growth, especially plant height. The analysis of variance between treatments significantly affected all observation variables, meaning that each treatment of the amount of mycorrhiza given could affect the growth and yield of tomato plants. One of the functions of leaves in tomato plants is to carry out the process of photosynthesis so that growth and development continue to increase (Zai et al., 2021). Photosynthesis produces energy in the form of ATP and NADPH compounds, where ATP is a source of energy for the metabolism of the plant’s body. The availability of nutrients such as P affects the formation of ATP. This is related to mycorrhiza in plants, which can increase the absorption of nutrients, especially phosphorus. Increasing the P content in plant tissue can accelerate cell division, especially in the meristem tissue in plants, which affects the growth and development of the plant leaves.

Judging from the results, the provision of mycorrhiza affects the weight of the fruit between treatments. The dose of 75 spores is optimal for increasing the growth and development of tomato plants, significantly increasing fruit weight. Mycorrhiza can increase nutrient absorption and maintain soil water levels that are good for tomato plant growth. This is also in line with (Mohammadi et al., 2020)  opinion that mycorrhiza can increase nutrient absorption. Plants given mycorrhiza are more resistant to drought because the roots of mycorrhizal plants recover quickly after experiencing a lack of water. Mycorrhizal hyphae can still absorb the soil’s pores when the plant roots can no longer absorb water. In addition (Franczuk et al., 2023) also explained that mycorrhiza can replace approximately 50% of phosphate needs, 25% of potassium needs and 40% of nitrogen needs because mycorrhiza can increase the absorption of N, P, K, Ca and Mg elements compared to organic fertilizers. From this incident, it can be explained that the absorption of nutrients assisted by mycorrhizal hyphae affects the weight of tomato fruit. The more mycorrhizal spores given, the better it is for the growth of tomato plants.

The observation results of tomato plant root infection with the treatment of different amounts of mycorrhizal spores at different times of application show that all tomato plant roots inoculated with mycorrhizal spores are infected, but have different infection values. The correlation test results between the Mean Percentage of Root Infections (%) and the average percentage of disease incidence (%) in the 7 WAP showed that R2 = 89.02%. This indicated that the higher the average percentage of root infections, the lower the disease incidence.

According to (Smith, 2009), MVA can cooperate with 80-90% of plant species. It also revealed that one type of plant that can cooperate or colonize with MVA is from the Solanaceae family. Tomato plant roots infected by mycorrhizae are characterized by the presence of external and internal hyphae, as well as vesicles. Mycorrhizal infection with 75 spores resulted in a negligible effect on the level of tomato wilt disease attack, where more spores were given and colonized the roots of tomato plants. The higher the percentage of root colonization by AMF, the smaller the percentage of disease attack intensity (Campo et al., 2020). Mycorrhiza uses carbohydrates and plant root exudates, thus creating an unsuitable environment for pathogens around the plant. In addition, mycorrhizal fungi can secrete antibiotics that can be toxic to pathogens.

The roots of tomato plants infected by mycorrhiza will experience morphological changes, namely the formation of lignin in the endodermis of the plant roots, so that it can be a barrier against pathogen attacks and tomato plants will increase their resistance to Fusarium oxysporium wilt disease (Pu et al., 2022). Other studies also explain that plants that are symbiotic with mycorrhiza will experience increased flavonoid content because stimulation occurs when plants are infected with MVA. Colonization will form in the roots, making the plants more resistant. In addition (Vierheilig and Bago, 2005) also explains that plants infected with MVA will experience lignification in the roots and parenchyma, where lignification is part of the cell wall defence against pathogen infection.

Ralstonia solanacearum is a bacterium that causes wilt disease in tomato plants; it also attacks other plants, such as chili plants, tobacco and potatoes. Bacterial attacks on tomato plants found in experimental fields are suspected of Psedomonas solanacearum bacteria, currently known as R. solanacearum. This soil-borne pathogen can survive in plant debris for a long time and spread quickly to injured plant parts. In addition, this bacterium infects its host through the roots during transplanting. (Boutaj et al., 2022) Also explained that attacks by R. solanacearum bacteria are also influenced by environmental factors such as high rainfall and high humidity, so pathogen growth and development cannot be suppressed optimally. The influence of the environment affects microbes such as mycorrhiza, which are less than optimal in suppressing attacks of bacterial wilt disease caused by R. solanacearum . This study showed that plants infected with mycorrhiza were still attacked by the R. solanacearum bacteria, as indicated by the brownish inner stem of the tomato.

Based on the results of the study, it is known that there is an interaction between the Treatment of the number of mycorrhizae and the application time. The best combination of growth treatments is M3T1 (75 Spores + seedling time). This can be seen in several parameters, such as plant height and number of leaves. A dose of 75 mycorrhizae spores is known to suppress bacterial wilt attacks caused by Ralstonia solanacearum by 21.38%. Root infection of tomato plants treated with M3T1 (75 spores + seedling time) has a high mycorrhizae infection rate of 53.78%.

The present study was supported by the University of Muhammadiyah Malang.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the author and do not necessarily represent the views of their affiliated institutions. The author is responsible for the accuracy and completeness of the information provided, but does not accept any liability for any direct or indirect losses resulting from using this content.
 
Informed consent
 
We did not use animals for this research.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the study’s design, data collection, analysis, publication decision, or manuscript preparation.