The Use of rhizobia and vesicular-arbuscular mycorrhiza to improve drought resistance of Kudzu (Pueraria phaseoloides (Roxb.)Benth.) in coastal land

A study was conducted to examine the association between Rhizobium and VAM fungi on the kudzu plant growth and response to drought stress in coastal dry land. The research was carried out in three phases: (1) germination of kudzu seeds; (2) multiplication of VAM and Rhizobia inoculums; and (3) inoculation of fungi and/or bacteria on plants grown at various soil moisture content. At the inoculation phase, the pot experiment was carried out for 8 weeks with a pot experiment arranged based on a 4 x 4 treatment factorial Completely Randomized Design with three replications to test the source of the inoculum and soil moisture content (25%, 50%, 75%, 100%). Observations were made on: (1) Rhizobial infection activity and nodulation; (2) VAM infection activity; (3) plant growth, and 4) population dynamics of rhizobacteria. The results showed that the rhizobia inoculation alone was not able to increase the bacterial infection activity and root nodulation, the VAM fungal infection, and plant growth. Inoculation with VAM fungi alone was able to stimulate infection activity of nodule-forming bacteria and root nodulation, VAM fungal infection activity, and plant growth. The combination of VAM fungal and rhizobial inoculation was able to stimulate fungal infection activity and growth of plant roots and shoots, but not always able to stimulate bacterial infection activity and root nodulation. Inoculation of VAM fungi alone was able to stimulate the plant growth rates even up to a drought stress level of 75%. This means that inoculation of VAM fungi alone was able to increase drought resistance and maintain the plant growth rates at high levels of drought stress.


Introduction
Indonesia, with a two thirds of its ocean surface larger than the mainland, almost all islands have coastlines of at least 80,000 km which makes it longest coast [1].Most of the longest coastline is coastal sandy land which is widely used in agricultural cultivation.However, this land has many limitations and becomes an obstacle for farmers to cultivate crops [2].This advantage faced by coastal sandy land which is a critical land due to low nutrient concentration, unfavorable microclimate (drought), high wind and soil erosion, and little soil-fertilizing microbial propagules [3].To reduce the level of wind soil erosion and improve soil quality, among others, by planting conservation crops, including kudzu.Pueraria phaseoloides, also known as tropical kudzu, is a vigorous, perennial climbing plant producing annual, stems up to 5 mm in diameter and 2 -10 metres long from a tuberous rootstock.These stems scramble over the ground, where they produce new roots at the nodes, and also twine into the surrounding vegetation for support [4].Kudzu is a pioneer plants which often grows wildly on degraded or critical lands, including sandy coastal land.Kudzu plants are leguminous plants which are widely planted for land conservation [5].Naturally, the kudzu plant can function as a cover crop that can protect top soil from wind or water erosion, increase soil organic matter, increase soil microorganisms, increase water content and soil air circulation, reduce the use of herbicides, and provide nutrients to support plant growth [6].
The crops are known to associate with a root nodule-forming bacteria (Rhizobium sp.) which give some benefits to nutrient cycling i.e. : atmospheric N2 fixing and play role as soil conditioner; soil Nitrogen enrichment; nutrient cycling; and increasing other nutrients availability.Root nodules are symbiotic organs capable of fixing and reducing N2 from the air into nitrogen compounds that can be utilized by leguminous plants.Symbiotic nitrogen fixation is a complex interaction between host 2 plants, the environment and Rhizobacteria [7].From the results of previous research [8], it is known that the success of planting kudzus can be increased by inoculating selected Rhizobia isolates.Rhizobia bacteria is very significant for the ecosystem as well as for increasing soil fertility and plant growth itself.Its ability to bind atmospheric Nitrogen will make the soil planted by kudzus more fertile, so that in the long run it will increase the growth of other cultivated plants.It was also found that the legume roots to be infected by Arbuscular Mycorrhiza (AM) in the rhizosphere.The presence of arbuscular mycorrhizae in the legume plant is very interesting, since it provides many advantages, including helping the absorption of P especially, increasing plant resistance to drought, increasing the availability of pathogens, and even producing phytohormones that stimulate plant growth [9].The ability to provide a key ecosystem services, including protection of leguminous plants against biotic and abiotic stress, is enjoyed by arbuscular mycorrhizal fungi (AMF) and plant growth promoting rhizobacteria (PGPR).In their experiments with AMF (Rhizophagus clarus) and/or PGPR (Bacillus sp.) inoculations, [10] concluded that the AMF and PGPR consortium had the potential to increase nutrient uptake by plants experiencing drought to a moderate level (30%) compared to plants that were not inoculated.This shows that AMF and PGPR inoculations, both individually and together, significantly improve plant tolerance to drought and increase nutrient uptake and plant growth.In other research, [11] was also found that Pueraria phaseoloides contributes to macronutrients (N, K, Ca, Mg, P, S) cycling and become an option of sustainable management.Considering the various contributions of kudzu to conservation and increasing soil fertility, this research was conducted to examine the effect of inoculation of Rhizobium bacteria and/or Vesicular Arbuscular Mycorrhiza (VAM) on the ability of plants to adapt to drought stress and increase plant growth.

Methods
The research was carried out in three phases: (1) germination of kudzu seeds; (2) multiplication of VAM and Rhizobia inoculums; and (3) inoculation of fungi and/or bacteria on plants grown at various soil moisture content.

Phase 1: Germination of kudzu seeds
After cutting the exocarp to break dormancy, the kudzu seeds were germinated in containers and grown until they were 2 weeks old.Sterile sand was used as a growth medium, and during the nursery process, watering is carried out 2 times a day, or if the media looks dry.The seedlings used for the experiment were those that already had true leaves and were two weeks old, as shown in Figure 1.

Phase 2: Multiplication of VAM and Rhizobia inoculum
This phase is carried out by separating bacteria based on cell shape, colony shape, aerobicity, elevation, edge shape and inner structure into Yeast Mannitol Agar (YMA) medium for Rhizobia and Luria Berthani Agar (LBA) for rhizobacteria for 2 days.Then grow it on slanted agar medium and in broth medium for 2 days.After 2 x 24 hours, the colonies obtained were pure, so the inoculum was propagated in liquid medium.Observations were made on: a.The effectiveness of Rhizobia infection, consisting of: total number of nodule; number of effective nodules; percentage of effective nodules (%); and nodule weight (g).b.The mycorrhizal infection activity, including: percentage of VAM infection (%), root fresh weight (g), root dry weight (g), and root length (cm) c.Population dynamics of rhizobacteria in soil which includes the number of rhizobial based on the type of inoculum and soil moisture.

Phase 3: Inoculation of fungi and bacteria on plants
This phase is a trial pot test and aimed to determine the effect of Rhizobia and VAM inoculums on the growth of shellfish plants at various moisture levels.This phase of the research was carried out for 8 weeks, with a pot experiment arranged based on a 4 x 4 treatment factorial Completely Randomized Design with three replications.The treatments tested were inoculations of Rhizobia, VAM, and Rhizobia-VAM and controls.The second factor is soil moisture content which consists of 25% of field capacity (75% drought stress), 50% f.c.(50% drought stress), 75% f.c.(25% drought stress), and 100% f.c.(no drought stress).Observations were carried out at weeks 8 including: plant length (cm), shoot fresh weight (g), number of leaves, leaf area (cm 2 ), and shoot dry weight (g).

Data analysis
Analysis of variance was carried out to test the effect of treatment on the parameters measured.If the value of F count > F table , then it means that the treatment has a significant influence on the observed response.If so, then a further test is carried out with the Duncan Multiple Range Test to obtain information about the treatment that gives the best response.

Rhizobial Infection Activity and Nodulation of Kudzu Plants
Kudzu plants are known to be symbiotic with root nodule-forming rhizobacteria, mostly from the Rhizobia group.The existence of this Rhizobia bacteria is very significant for the ecosystem as well as for increasing soil fertility and plant growth itself.Its ability to bind air Nitrogen, in the presence of bacteroid, will make the soil planted with kudzu plants more fertile so that in the long run it will increase the growth of other cultivated plants.Longitudinal sections of kudzu root nodules show a generally round nodule shape, sometimes having a stalk or not.Effective nodules are marked red when sliced due to the presence of bacteroids (Figure 2).In this study, bacterial infection and nodulation activity could be observed based on the total number of nodules, the number of effective nodules, the percentage of effective nodules and the total nodule weight, as presented in Figure 3 and Table 1 below.

Figure 3. Effect of inoculation on VAM infection activity
Figure 3 demonstrates that plants inoculated with VAM had the highest number of nodules, and Table 1 show that 79.64% of those nodules are functional.In contrast, plants inoculated with Rhizobia and VAM-Rhizobia exhibited almost no nodules and effective nodules.No-inoculation treatment resulted in a low number of nodules and the effective ones (Table 1).There was no interaction between microbial inoculation and soil moisture treatments.Moisture content treatment did not affect the activity of bacteria.Instead, the inoculation treatment significantly affected the infection and nodulation activity.The inoculation treatment significantly affected the infection and nodulation activity.VAM inoculation alone gave the highest results in the total number of nodules and the effective ones, percentage of effective nodules, and nodules weight.In general, VAM fungi application can increase the number of nodules due to the presence of VAM fungi which can absorb nutrients, especially phosphorus, through the spread of the formed hyphae, which can increase the absorption of nutrients for the formation of root nodules.The presence of VAM fungi also helps the roots of host plants expand the absorption area of root hairs through mycelium formation around the roots and increase nodulation and N2 fixation by Rhizobia.In coastal sandy soil, the VAM fungi can supply Rhizobia propagules in the soil to have the best effect on forming effective root nodules.VAM can make soil conditions more dynamic by influencing the growth of Rhizobia bacteria indigenous in coastal sandy soil, so that root nodule formation activities will run smoothly.The entry of Rhizobia bacteria into the roots is assisted by the presence of VAM inoculum so that compatible bacteria will quickly enter and develop in the plant roots.As a result, it accelerates bacterial activity to form root nodules, and the active period of root nodules is longer.Meanwhile, variations in moisture content did not affect the activity of the bacteria, which means that the total number of nodules and the adequate nodule number relatively did not differ in all soil moisture treatments, even with decreasing soil moisture tends to increase in number.

VAM infection activity
All roots in all treatments were infected with VAM.Vesicle and arbuscle formations could be seen in the cortical cells of kudzu roots that had been exposed to VAM or had not.The microscopic appearance of vesicle and arbuscle structures in roots is seen in Figure 4.It is believed that they are from the genus Glomus or Gigaspora based on the types of vesicles that were discovered.

Figure 4. Microscopic appearance of vesicles (V) and arbuscles (A) in root
The study's findings revealed that varied amounts of VAM mycelium were discovered in the kudzu root cells across all treatments.The presence of vesicles and arbuscles from VAM will help plants take up nutrients.Even in the absence of inoculation, kudzu roots were infected with VAM (Table 2).In fact, VAM inoculation treatment, with or without Rhizobia, produced roots with a larger volume (Figure 3).
Leguminous plants' symbiotic association with mycorrhiza fungi (arbuscular mycorrhiza fungi or vesicular arbuscular mycorrhiza fungi) may ultimately boost root size and efficiency, leaf area index, and biomass under drought circumstances [9].As a result, plants inoculated with VAM-Rhizobia or VAM alone have a higher VAM infection rate, generate heavier roots (Tabel 2), and better plant growth (Table 3).There was no interaction between the soil moisture treatments and microbial inoculation.The amount of moisture has little effect on fungal activity.Meanwhile, the inoculation treatment had a considerable impact on infection and nodulation activity.Only a few VAM infected roots were given in the treatment without inoculation, allowing the plants to spread their roots to take up water and nutrients.The large percentage of infected roots in the VAM and VAM-Rhizobia inoculation treatments will increase the area of water and nutrient absorption as well as supply nourishment for rhizobacteria, enhancing the efficacy of root nodulation.The high percentage of infected roots in plants inoculated with VAM or Rhizobia-VAM causes the plants to meet their water and carbon needs, requiring little energy to extend.

Plant Growth
As evidenced by plant growth, the availability of nutrients, particularly Phosphorus from VAM activity and organic Nitrogen from Rhizobia activity, can meet the plant's needs for growth and development.The inoculation treatment has a significant impact on plant development.At 3 months after transplanting, the control treatment generated numerous leaves but tiny in size, whereas the VAM-only or VAM-Rhizobia inoculation treatment produced a significantly large number of leaves and broad leaves, as shown in Figure 5. VAM inoculation also results in plants with longer stems.As a result, VAM inoculation, with or without Rhizobia, can result in superior plant development compared to other treatments (Table 3).

Figure 5. Effect of inoculation treatments on plant growth
There was no interaction between microbial inoculation and soil moisture treatments, and the moisture content has little effect on fungal activity (Table 3).Interactions between treatments were only discovered in terms of their effect on shoot dry weight (Table 4).There was an interaction between bacterial inoculation and fungal VAM treatment on dry shoot weight.The Table shows that at a moisture content of 25%, the VAM inoculum alone gives good results compared to a moisture content of 25% at other types of inoculum.Drought stress has a number of effects on plant life; for example, a shortage of water reaching the roots reduces transpiration and increases oxidative stress.Drought stress slows plant growth through changing enzyme activity, ion absorption, and nutrient digestion.Plant drought tolerance may be due to roots and the fungi's extra-radical hyphae examining a large volume of soil [9]; [12].Figure 6 shows that plants from all treatments grown on soil with 75% drought stress produced enlarged roots as a result of the production of mycorrhyzal hyphae.This condition allows plants to survive in limited ecosystem conditions.

Population dynamics of rhizobacteria
Many plants in natural environments can grow in nutrient-poor soils by collaborating with microbes for mutual benefit, a phenomenon known as 'symbiosis'.One of the most common and significant symbiotic connections is arbuscular mycorrhiza, which is formed by interactions between soil fungus and more than 80% of terrestrial plants.Even when the soil conditions are water stressed, arbuscular mycorrhiza can stimulate the rate of photosynthesis and photosynthate transport to roots, the production of phytohormones and the exudation of organic acids from roots, as well as membrane permeability to nutrient pathways [13], [14], [10].
As seen in the above Figure 6, VAM infected all plant roots from all treatments, which could boost root system growth.The presence of mycorrhizal hyphae in these roots stimulates rhizobacteria growth in the rhizosphere, and vice versa.Figure 7 shows the population dynamics of rhizobacteria in the rhizosphere of kudzu plants based on microbial inoculation treatment and soil water content.The high number of rhizobacteria population at the early stage of growth shows that plant roots serve as a source of rhizobacteria capable of protecting plants from drought stress, as indicated by the highest number of populations in the treatment of drought stress up to 75% (25% moisture content).The number of rhizobacterial populations increases in the VAM and VAM-Rhizobia inoculation treatments, particularly at the early stages of plant development, indicating that the inoculum can stimulate rhizobacterial growth in the rhizosphere.
In his research [15] stated that foxtail millet plants grown on dry land will naturally associate with rhizobacteria which are highly resistant to drought stress and stimulate plant growth.Plant roots in symbiotic relationships with microorganisms produce exudates that improve the physicochemical properties of the surrounding soil while also preserving the function and structure of the microbial community near plant roots [16], [17].

Conclusion
x Rhizobia inoculation alone could indeed increase the activity of these bacterial infections and root nodulation but had not improved root and plant growth.x VAM inoculation alone has not been able to increase the activity of the fungal infection.
However, it can significantly increase the activity of Rhizobia infection and root nodulation, and maintain plant growth, even when the plants experience drought stress of up to 75%.x Inoculation of VAM with Rhizobia increased VAM fungal infection, increased Rhizobia infection activity and nodulation, and increased plant resistance to drought.x Rhizobium inoculation alone and with VAM can affect the presence of the Rhizobacter population in the soil for up to 6 weeks.A 25% moisture content can maintain the Rhizobacter population for up to 6 weeks in the soil.

Figure 2 .
Figure 2. Morphological photograph of kudzu root nodule (a); cross section of nodule (b); bacteroid in the nodule (c)

Figure 6 .
Figure 6.Root condition of kudzu under inoculation treatment and 25% soil moisture stress

8 Figure 7 .
Figure 7. Population dynamic of rhizobacteria based on type of inoculum (a) and soil moisture content (b)

Table 1 .
Effect of inoculation and soil moisture content on rhizobial infection activity and nodulation at eight weeks after transplanting Values followed by different letter in the same column indicate the significantly differences according to the F Table5%, (+) there is an interaction.f.c. is field capacity, ds = drought stress.

Table 2 .
Effect of inoculum type and moisture content on VAM fungal infection at eight weeks after transplanting Notes : Values followed by different letter in the same column indicate the significantly differences according to the F Table 5%, (+) there is an interaction.f.c. is field capacity, ds = drought stress.

Table 3 .
Effect of inoculum type and moisture content on plant growth at eight weeks after transplanting Values followed by different letter in the same column indicate the significantly differences according to the F Table 5%, (+) there is an interaction.f.c. is field capacity, ds = drought stress.1255 (2023) 012006 Notes :

Table 4 .
Effect of inoculum type and moisture content on shoot dry weight at eight weeks after transplanting : Values followed by different letter in the same column indicate the significantly differences according to the F Table5%, (+) there is an interaction.f.c. is field capacity, ds = drought stress. Notes