Exploration of Vesicular Arbuscular Mycorrhizae (VAM) chili plants in various field types in Indonesia: a case study in Kolaka Regency, and their relationship with Fusarium oxysporum wilt disease

The symbiotic relationship between mycorrhizal fungi and plant roots provides benefits to the host plant. Fusarium wilt, caused by Fusarium oxysporum, is the most common disease on chili (Capsicum annuum), resulting in production losses of up to 50%. Until now, efforts to control wilt disease still rely on chemical control which has a negative impact on the environment.The purpose of this study was to examine the existence and number of Mycorrhizal spore morphology surrounding the roots of chili plants in three different types of fields in Kolaka Regency, as well as their relationship to Fusarium wilt. Plants were sampled from numerous chili farms in Kolaka Regency for this study, and the samples will be identified at the Plant Disease Laboratory, Faculty of Agriculture, Hasanuddin University, Makassar.The proportion of infection, the amount of spores in the soil, and the incidence of fusarium wilt in chili were all measured. Mycorrhizae were found to infect chili roots and increase the amount of spores in three separate fields of chili plants. This demonstrates a link between mycorrhizae and chili roots, as well as the presence of spores in the soil and the kind of spores, Glomus sp. and Gigaspora sp.The interaction between mycorrhizae and Fusarium oxysporum disease in chili plants, namely suppressing the acceleration rate of Fusarium oxysporum disease is said to occur but is considered low. Different field types significantly affect the characteristics of mycorrhizae and the growth of chili plants, especially those that are attacked by Fusarium oxysporum. The field type with the least spores were found in Sani- Sani Village, Samaturu District, Kolaka Regency.


Introduction
Chili (Capsicum annuum L.) is a commodity that has a high economic value [1].Chili production in Kolaka until 2020 reached 7,321 quintals with a total plantation area of 61 hectares [2].One of the main factors in chili cultivation is the soil types.Pest and disease management is another important aspect in the success of chili growing.High humidity, ambient temperature, and the increased dissemination and development of plant pests and diseases are drivers of chili production performance [3].Mycorrhizae are a type of symbiotic mutualism between plants and fungi that infiltrate the root cortex during plant development.
Mycorrhizae is a form of symbiotic mutualism between plants and fungi that colonize the root cortex during the growth period of the plant.Several studies have shown that the application of arbuscular mycorrhizae alone has a higher control effectiveness compared to the combination with endophytic 1230 (2023) 012106 IOP Publishing doi:10.1088/1755-1315/1230/1/012106 2 fungi.Arbuscular mycorrhizae were able to colonize onion roots well, prolong the latent period, reduce disease incidence and significantly increase plant growth [4].Exploration of mycorrhizae from various places and the application of mycorrhizae to plants as biofertilizer technology is important for research.So that we get several mycorrhizal genera and get a suitable mycorrhizal genus for plants [5].
The diverse population and composition of VAM are not only influenced by environmental factors such as temperature, soil pH, soil moisture, phosphorus and nitrogen content and heavy metal concentrations are also affected by the characteristics of the plant species.The aim of the study was to determine the presence and number of spores and to determine the morphological characteristics of arbuscular mycorrhizal fungi (AMF) spores around the roots of chili plants in the three different soil typesin Kab.Kolaka and its relation to Fusarium oxysporum wilt.

Material and methods
Soil samples from around the roots of chili plants were collected from three investigation places in Popalia Village and Puulemo Village, Baula District, and Sani-Sani Village, Samaturu District.Aquades, H2O, 10% KOH solution, 1% HCl solution, lactophenol blue, spirit, tissue, aluminum foil, gauze, and label paper were used.The technique utilized in this study is a descriptive exploratory approach with purposive sampling.

Soil sampling
Soil sampling was conducted in each root area of chili plants.Before taking soil samples, measurements were taken from 0-10 cm from the tree, then the soil was excavated with a depth of 0-20 cm from the ground surface and then put in the sample envelope.

VAM spore isolation
Isolation of VAM spores used a combination method of pouring a wet filter technique [6] and conducted in months 1, 2 and 3 with a wet filtering technique.Furthermore, the number of spores was observed on a line of filter paper under a microscope with a magnification of 40x100 times or it could also be observed on a Haemacytometer.

Identification of VAM spore morphology
Identification of arbuscular mycorrhizal fungi was conducted based on the similarity of spore morphological characteristics including the color and shape of spores.According to Brundett (1991) [7] the identification of arbuscular mycorrhizal fungi was conducted based on the similarity of spore morphological characteristics including color and shape of spores.

Staining of root tissue
Root coloring was conducted to see the structure of the fungus in the plant root tissue so as to facilitate the observation of colonization by biocontrol agents.Root staining was conducted using 5% Acid fuchsin dye.The onion roots are cleaned and put in a glass bottle.Chili roots are then processed sequentially, namely clearing and staining.Clearing was done by soaking the roots in a 10% KOH solution and then washing them with running water until the roots were clear.The next step is staining the roots by soaking them in a dye solution for 12 hours [8].

Percentage of VAM infections (%)
The percentage of VAM infection was conducted by taking a sample of 10 pieces of chili plant roots, painted with lactophenol blue, then observed with a microscope the percentage of infection with the formula: Percentage of VAM Infection =

Number of spores
The observation of the number of spores was conducted using a wet filtering technique and then observing the number of spores on the grid lines on filter paper that had been made with a microscope with a magnification of 40x100.Furthermore, the spore count was conducted to determine the density of the spores.Spore density is the number of spores per soil sample analyzed.Spore density is calculated by the formula: Spore density = number of spores per amount of soil analyzed.

Observation of the incidence of fusarium wilt disease in chili plants
Observations observed were the incidence of disease.Disease incidence is the ratio of diseased plants to healthy plants.The incidence of disease was determined based on the Vanderplank formula [9]: n Disease Incidense = --------x 100% (2) N Information: n: Leaves that show symptoms of disease

Results and discussion
This research was conducted in three separate chili fields: Popalia Village, Puulemo Village, Baula District, and Sani-Sani Village, Samaturu District, Kolaka Regency.The following sub-chapters cover the features and research results of the three fields:

Chili farm in Popalia Village, Baula District, Kolaka Regency
Popalia Village, Baula District, Kolaka Regency's chili field has never been planted with anything other than chili and will be converted to oil palm plantations.Because the field owner is focusing on turning its purpose to oil palm field and other enterprises such as boarding homes and food stalls, the observed field appears to be abandoned or badly managed.Maintenance is provided in the form of goat dung, liquid fertilizer, and NPK Mutiara.Lice, base rot, and fusarium wilt are common pests.The Rovral brand of fungicide was used to combat root rot, and the Nordox brand was used to control Fusarium.There has been no research on the content of microorganisms in this sector.Chili plants were studied in four replications in Popalia village, with three plants showing signs of fusarium infestation and one plant seeming healthy.

Chili farm in Puulemo Village, Baula District, Kolaka Regency
The second land to be explored is located in Puulemo Village, Baula District, covering an area of 0.5 ha.The variety of chili used is Dewata.The age of chili plants at the time of observation was 2.5 months with the number of harvests 2x a week.Pak Yohanes's land is not only planted with chili, but also tumpengsari with long beans and cucumber.The soil type is almost the same as the soil at the first location, namely red-yellow Podzolic soil with a temperature at the time of observation 26 o C, humidity 90%.The soil texture in this area is clayey loam with a pH of 6.3 (slightly acidic).The texture of the soil in this village in the dry season is cracked dry ground surface and during the rainy season it resembles clay.Therefore, the treatment given by farmers so that chili can be planted is by giving dolomite lime and using mulch.
The pests that often attack the chili in Puulemo village are base rot and fusarium wilt.Root rot was controlled with the Rovral brand of fungicide and Fusarium was controlled with the Nordox brand.In this land there has been no research on the content of microorganisms in it.The observation of chili plants in Puulemo village was observed for 4 replications consisting of 3 plants that indicated symptoms of fusarium attack and 1 plant that looked healthy.The chili fields observed in this village have a dusty clay texture with a slightly acidic pH, low and very low C, N levels.C/N levels are high, P and K are very low and low.The treatment given was only planted on the edges of the shrimp pond.There is no disease prevention and it is only planted as a side crop.The history of cultivation on this land is that of other vegetables such as tomatoes and green eggplant.

Percentage of infections (%)
Checking chili plant root infection in each plant was conducted by taking 20 pieces of tertiary roots.The calculation was done by counting the number of mycorrhizal-infected and non-infected root parts in each root.The mycorrhizal proportion was the total number of mycorrhizal parts divided by the total number of roots observed, multiplied by 100%.This method is a modification of the method of Giovannetti and Mosse [10].The visualization of mycorrhizal-infected chili roots is shown in Figure 1 and Figure 2.  In the picture above, it can be seen that vesicles are on the inside of the root, round in shape, this is in accordance with the research whose conducted by [11].The vesicles can be found both inside and outside the cortical parenchyma layer.This is a swelling of the hyphae contained within and between the cortical cells, the number of vesicles will increase as the mycorrhizae get older and plant.Figure 8 also shows internal hyphae, which look like branching threads that are inside the roots.
The result on three fields in Popalia, Puulemo, and Sani-sani villages, particularly the roots of chili plants, were 100% infected with mycorrhizae.This is in accordance with the opinion of [11] that mycorrhizae are fungi that live in symbiosis with plant roots.Table 1 shows the proportion of mycorrhizal infections in chili roots.Isolation and Identification of VAM Spores Soil isolation from three sampling locations produced several types of VAM spores in the chili plant rhizosphere through a wet and stratified filtering process and could be observed under a microscope.Identify the type of spore isolated based on morphological characteristics which include shape, size, color, wall, and surface texture of the spore.The shape of mycorrhizal spores can be used as a feature to determine the types.Based on the results of exploration, the data on the number of spores found in various fields of chili plants are attached in table 2. In addition, in this study also found two types of Glomus sp. and Gigaspora sp.just.The results of the identification and number of VAM spores (the number of spores per 100 g of soil) in three villages (Puubunga, Puulemo and Sani-Sani) from sampling can be seen in table 3. The table above shows that the spores of Glomus sp. and Gigaspora sp. in the three types of explored field have different values.On field in the village of Popalia, the abundance of Glomus is very much compared to Gigaspora sp.Likewise on the second field in the village of Puulemo.In the third field in the village of Sani-Sani, the ratio of the abundance of Glomus and Gigaspora species was also the same, that is, the numbers were very far apart.This shows that Glomus has a fairly good level of adaptation in various environmental conditions.Soil types, nutrient availability and the history of planting in the three villages are different.Certainly, this had an effect on the growth of mycorrhiza in each chili field in the three villages.This is in accordance with the existing theory that the availability of nutrients affects the growth and spread of mycorrhizae [12].
In the percentage of chili root infection, although all of them were 100% infected with mycorrhiza, this was different from the number of VAM spores.The low number of spores is thought to be due to environmental conditions that are not suitable, optimal, and less compatible in supporting the growth and development of VAM spores and the possibility of a lack of maintenance from the owners of these lands.According toMuhibuddinA,et.al.(2007) [13] there is a significant correlation between rainfall, soil temperature, soil N, P and K content on the development of fungal spores.The type of Glomusand Gigaspora are found in all three which have the structure of clay loam and dusty clay, but the amount varies.
Soil dominated by the clay fraction is a predictable condition for the development of Glomus spores, meawhile in sandy soils the Gigaspora was also found.In sandy soil, the pores of the soil formed are larger than in clay soil and this condition is thought to be suitable for the development of Gigaspora spores which are larger than Glomus spores.In this study, the number of spores found in Sani-Sani village with the type of dusty clay had the least number of spores.The number of spores in the three chili fields that have been explored can be assessed as lacking.
Research by [12] explained that the number of mycorrhizal spores in legumes on intensive tillage without the addition of N fertilizer, has the highest number of mycorrhizae which are 4,078 spores and the addition of a higher dose of N, the number of spores decreased by 1,124 spores, at the highest dose (200 g), the number of spores was 941 spores.

The incidence of chili plant diseases
The observation of chili plants was conducted in three locations, in Popalia Village and Puulemo Village, Baula District, and Sani-Sani Village, Samaturu District, Kolaka Regency.The three chili fields all have chilies that are indicated by the symptoms of Fusarium wilt attack.The selection of exploration location points was based on the direction of the extension worker in Baula sub-district and the head of horticulture at the Food Crops and Horticulture Office of Kolaka Regency.This location was chosen because only the chosen sub-district is growing chili plants.Based on observations of the symptoms of Fusarium wilt in Popalia Village, Puubunga Village, and Sani-Sani Village, the most common symptoms found in chili during observation were in Popalia Village, Baula sub-district.Symptoms include yellow spots on the edges to the entire leaf surface.The highest incidence of Fusarium wilt was found in the third location, namely in Sani-sani Village, Samaturu District.Meanwhile, the symptoms of Fusarium wilt at the second location in Puubunga village, Baula subdistrict, were low to moderate.It be caused by several factors such as soil type, pH, nutrient content and others that affect the growth of fusarium wilt and mycorrhizal disease itself.The results of the calculation of disease incidence are presented in table 4. The disease incidence percentage from the chiliplant which were explored in the three locations are different.This was because, apart from being located on land with different conditions.In addition, when taking plant samples and bringing them to the observation site, they are not close to the exploration site.This is in accordance with [14] who revealed that disease development is closely related to the incubation period, pathogen virulence, environmental conditions, and susceptible host plants.These factors can affect the percentage value of the incidence of wilt disease symptoms that attack chili plants even though they are in the same field.
According to the results of observations made by the author, some of the wilted chili plants in the three locations were considered to be attacked by Fusarium wilt, some were considered to be attacked by virus and lack of nutrients.This is based on the opinion of Field Agricultural Extension during joint observations with the author.The assumption about being attacked by Fusarium wilt is because there are several plants that show these symptoms, as well as wilt which is considered to be infected with a virus.As for the chili plants which are considered nutrient deficient, this is characterized by the many plants where some of the leaves are yellowish and the fruit is small.In addition, the conditions around the plantations are not well maintained.
In this study, to ascertain whether the chili plants observed were attacked by Fusarium wilt, further research is needed in the laboratory to determine whether those attacking chili plants were actually attacked by Fusarium pathogens or other pathogens.[15] states that if antagonistic agents dominate plant roots, the growth of the pathogen is inhibited so that it does not thrive.According to several theories that have been mentioned, mycorrhizae can prevent the incidence and severity of wilt disease caused by Fusarium fungi as in the study of Ni Putu, et.al [1] which showed that the application of VAM spores of 100 g, 200 g, and 300 g VAM to cayenne pepper plants was infected with fusarium showed a significant effect on the positive control.The control produced less fruit because without VAM inoculation the plants became more susceptible to fusarium wilt.The documentation of plants showing symptoms of fusarium wilt attack is attached in the photo below.Environmental conditions that support plant growth and are less supportive for the development of pathogens slow down the incubation period, so that pathogens take longer to infect plants according to Alfizar, et.al (2011) [16].Plants that were not inoculated with VAM had discolored xylem lengths that were significantly different from those that were inoculated with VAM.The faster the incubation period, the faster F.oxysporum infects the plant xylem so that the longer the xylem is discolored [17].VAM symbiotic with plant roots by colonizing apoplast and cortical cells [18].VAM uses carbon produced by photosynthetic plants so that pathogens do not get a food source.Thus, the growth of pathogens is inhibited so that plants become more resistant to pathogen attacks [19].
Therefore, even though the chili plants in the three exploration villages had mycorrhizal infected roots and the soil contained mycorrhizal spores, the chili plants could still be said to be infected with wilt disease which was assessed as viral wilt, nutrient deficiency, and Fusarium wilt.[20] added that plants colonized with VAM suffered little damage and the development of pathogens was hampered because VAM was able to strengthen the morphological and physiological characteristics of plants by changing the chemical composition of plant tissues.This theory proves that the presence of mycorrhizae can inhibit fusarium wilt infection, but what has happened in the fields that have been explored shows the opposite.The possibility that can occur besides fusarium wilt is a lack of nutrients and maintenance of chili plants.Lack of nutrients can also cause yellowish wilt in plants [21].

Conclusion
This study concluded that the results of observations and observations made in three villages that had chili fields, the mycorrhiza species that were abundant in chili cultivation areas were dominated by Glomus sp.Although there are also types of Gigaspora too.
The interaction between mycorrhiza and Fusarium oxysporum disease on chili plants, namely reducing the rate of acceleration of Fusarium oxysporum disease can be stated to occur but is considered low based on observations on the number of spores and disease incidence.Differences in land types have a significant effect on the characteristics of mycorrhiza and the growth of chili plants, especially those attacked by Fusarium oxysporum.The type of soil on the third field, namely Sani-sani village, has chili plants with the highest percentage of Fusarium wilt disease.

Figure 1 .
Figure 1.The picture shown by the arrow is a vesicle of mycorrizhae present in the roots of the chili plant

Figure 2 .
Figure 2. The picture shown by the arrow is an arbuscule (a collection of hyphae) mycorrhizae found in plant roots

Figure 3 .
Figure 3. (a) Chili plants assessed to be attacked by Fusarium wilt, (b) Chili plants assessed for virus attack, (c) Chili plants which are considered to wilt yellow due to a lack of nutrients, (d) Chili plants which are considered quite healthy.

Table 1 .
Percentage of mycorrhizal infections in chili roots.

Table 2 .
Number of Spores per 100 grams of soil on exploration field in Kolaka Regency.

Table 3 .
Data on the number of Glomussp and GIgasporasp spores.In each type of field explored.

Table 4 .
Percentage of Fusarium wilt infection in chili in the three exploration fields.