The effectiveness of Arbuscula Mycorrhyza Fungi stater with different fortification in increasing the productivity of Clitoria ternatea on saline soil

. Saline soil has a high salt content and low productivity, so it is rarely used as agricultural land or forage land for animal feed. Arbuscular Mycorrhizal Fungi (AMF) is a soil fungus that can help plants absorb water and nutrients so that it can help increase plant growth and productivity. This study aimed to evaluate the effectiveness of AMF in increasing the productivity of Clitoria ternatea on saline soil. The research used a completely randomized design (CRD) with a factorial pattern of 5 x 3 with 8 replications. The results showed that the interaction effect of AMF treatment and saline soil was significant (P<0.05) for all variables. This study concluded that saline soil increased the proline content of Clitoria ternatea leaves, and reduced the amount of leaf chlorophyll. AMF could help Clitoria ternatea to overcome saline stress and reduce the use of fertilizer doses by 50%. The addition of AMF increased the total vertical length gain, canopy dry weight, percentage of root colonization, and root dry weight. The type of AMF product IPB 2 gave the best results.


Introduction
Saline soil is a sub-optimal soil that has high salt content and low productivity, so it is rarely used as agricultural land or forage land for animal feed.The results of the research of Orndorff et al. [1] showed that the Na content exchanged at 1.62 cmol/kg in saline soils made it difficult for plants to absorb water, resulting in low turgor pressure.Munns [2] stated that salinity reduces the ability of plants to absorb water, causing a decrease in growth speed, the same thing was conveyed in the results of Lee et al. [3] increased salinity decreased the growth of paspalum grass.
AMF is a fungus that forms a symbiotic mutualism with host plants, in helping plants absorb water, both macro, and micronutrients so that they can help increase plant growth, productivity, and quality [4].In addition, AMF is effective in increasing the resistance of host plants to salinity stress.Karti [5] stated that the application of AMF can increase the production of the dry weight of the canopy of Chloris gayana in saline soil.
Clitoria ternatea is a type of plant with a relatively good level of tolerance to drought stress [6].Kalamani et al. [7] reported that Clitoria ternatea has crude protein ranging from 14-20%.The dry matter production of Clitoria ternatea under optimal conditions is 30 tons/ha/year [8].The degree of adaptation of Clitoria ternatea to salinity stress has not been reported.Further in-depth studies are needed on the tolerance level of Clitoria ternatea in a saline environment and efforts to increase its tolerance under saline stress conditions through AMF inoculation.AMF inoculation on Clitoria ternatea under moderate and high salinity stress is expected to be effective in increasing plant adaptation to support its production Further evaluation is needed on the effectiveness of different fortified AMF starters in salinity-stressed Clitoria ternatea hosts.The purpose of this study was to evaluate the effectiveness of AMF products produced by the Agrostology Laboratory of the Faculty of Animal Science, IPB University in increasing the productivity of Clitoria ternatea on saline soil.

Materials and methods
This research was carried out from September 2021 -April 2022.Plant maintenance was carried out in the Greenhouse of the Agrostology Field Laboratory of the Faculty of Animal Science, IPB University.Further sample analysis was carried out at the Laboratory of the Faculty of Animal Science, IPB University.

Preparation of planting media
The study began by preparing planting media, namely control soil and saline soil.Medium and high salinity soils were obtained from the coast of Ciparage Jaya Village, Karawang, West Java, with electrical conductivity (EC) content of 6.1 ds/m (medium saline soil) and 8.4 ds/m (high saline soil) [9].The soil is sifted and cleaned of impurities, then weighed 5kg/polybag.Each planting medium was added with manure according to the treatment dose and homogenized.Details of fertilizer doses are presented in Table 1.

Planting and AMF inoculation
Clitoria ternatea seeds were sterilized with the active ingredient Sodium Hypochlorite for 7 minutes, then neutralized with distilled water.Scarified seeds were sown on soil media until 7 days after sowing.The growing Clitoria ternatea seedlings were transferred to polybag media, two seeds each, and 20 grams of AMF/polybag were inoculated according to the treatment.After the seeds are 2 weeks after planting (WAP), the selection of seeds is carried

Data collection
Plant height data was measured every week using a ruler.Clitoria ternatea plants from different AMF inoculation on various types of saline soil were harvested at the age of 9 WAP by cutting the base of the plant by separating the crown and roots.Dry biomass of plants and roots was measured at the age of 9 WAP through an oven at 60 o C until the weight was stable.Leaf chlorophyll data was measured using the TYS-B Digital Chlorophyll Meter Analyzer (SPAD).Measurement of leaf proline levels refers to the method developed by Bates et al. [10].AMF colonization data on the roots of Clitoria ternatea were measured using the [11] modified [12].

Data analysis and experimental design
The study was designed with a completely randomized design (CRD) with a 5 x 3 factorial pattern with 8 replications.Factor A is the difference in the type of AMF which consists of 5 treatments, namely without giving AMF (F0), commercial/superior AMF as a comparison (FU), AMF fortified AB Mix 1000 ppm (EM1), AMF fortified AB Mix 2000 ppm (EM2) and FMA fortified AB Mix 3000 ppm (EM3).Factor B is the difference in soil salinity consisting of 3 treatments, namely control soil (T0), medium saline soil (T1) and high saline soil (T2).The data obtained from the study were analyzed using analysis of variance or analysis of variance (ANOVA).If it shows a significantly different effect, then the analysis is continued by using Duncan's Multiple Distance test to determine the difference between treatments.Data processing was carried out using the IBM SPSS Statistics 24 program.

AMF colonization of Clitoria ternatea roots in various saline soil types
Based on the results of the analysis of the variance of different types of AMF starters on soils with different salinities, the results were significantly different (P<0.05) on AMF colonization, in general the products EM1, EM2, and EM3 had the same colonization consistency both at T0, T1, and T2.For FU, the level of colonization was good on control soil however, it was significantly (P<0.05)lower than EM3 on high saline soil.These results indicate that the staters EM1, EM2 and EM3 have a strong symbiosis with the host plant, especially in conditions that are very stressed by salinity.When plants are exposed to high salinity, the percentage of root colonization increases.This is because AMF is needed by plants and AMF is still able to survive in conditions stressed by salinity.In line with the results of Karti et al. [13] research that AMF is very necessary for plants to overcome saline conditions because it can help plants absorb water and nutrients.The presence of AMF colonization in F0 was caused by indigenous AMF.The presence of AMF colonization in F0 was caused by indigenous AMF can be seen in Table 1.

Increase in vertical length of Clitoria ternatea different amf inoculation results in saline soil
Based on the results of the analysis of the variance of different types of AMF starters on soils with different salinities, the results were significantly different (P<0.05) on vertical length increase of Clitoria ternatea with the pattern that AMF inoculations of FU, EM1, EM2 and EM3 were effective in increasing plant vertical length on saline soils.Different results were obtained in plants without AMF that the increase in salinity dose was significantly different (P<0.05) from the increase in vertical length.These results indicated that inoculation of AMF on saline soil was effective in increasing plant adaptation to vertical length growth of Clitoria ternatea.Karti et al. [13] explained that AMF is needed to help plants overcome saline conditions, host plants that are in symbiosis with AMF can produce phosphatase enzymes that can release phosphorus elements bound by soil colloids so that the absorption of phosphorus nutrients can be increased by plants.The increase in vertical length of Clitoria ternatea from different AMF inoculations in various soil types can be seen in Table 3.

Clitoria ternatea canopy dry weight production with different AMF inoculation results in saline soil
Based on the analysis of the variance of the different types of starter AMF on soils with different salinities, the results were significantly different (P<0.05) on the dry weight of the canopy.The dry weight production of AMF FU, and EM2 inoculated shoots on high saline soil was significantly (P<0.05)better than on normal/non-saline soil.AMF supports the production of canopy dry weight better on medium and high saline soils.The canopy dry weight values of FU, and EM2 on high saline soils were significantly (P<0.05)higher and equal to T0F0.This shows that AMF is very effective in helping plants to overcome salinity stress, AMF is very effective in providing nutrients for plants even though the fertilizer used is 50% dose.According Mansur et al. [14] mycorrhizal fungi can absorb nutrients such as P, N, K, Zn, Cu, and Fe in saline soil conditions.The dry weight of the canopy of Clitoria ternatea aged 9 weeks can be seen in Table 4.

Root dry weight
Based on the results of the analysis of further tests, the dry weight of the roots of Clitoria ternatea, the results of different AMF inoculations on saline soils showed significantly different results (P<0.05) with the pattern that AMF inoculations of FU, EM1, EM2, and EM3 were effective and stable in increasing root dry weight on saline soils.Different results were obtained in plants without AMF that the increase in salinity dose was significantly different (P<0.05) on root dry weight.These results indicated that inoculation of AMF on saline soil was effective in increasing plant adaptation to root dry weight of Clitoria ternatea.
The high root dry weight in FU, EM1, EM2, and EM3 treatments was due to the high percentage of root infection in these treatments.The high root dry weight in AMF-treated plants was in line with the high average vertical length gain and canopy dry weight production.This shows that AMF is very helpful for plants to grow and cope with saline stress.Root dry weight of Clitoria ternatea from different AMF inoculations in various saline soil types can be seen in Table 5.

Leaf chlorophyll
Based on the results of the analysis of further tests, the amount of chlorophyll in the leaves of Clitoria ternatea, the results of different AMF inoculations on saline soils showed significantly different results (P<0.05) with the pattern that AMF inoculations of FU, EM1, EM2, and EM3 were effective and stable in increasing leaf chlorophyll on medium saline soils.In high-dose saline soil all AMF treatments the amount of chlorophyll decreased, this was because the plants experienced saline stress at quite extreme levels.The high leaf chlorophyll in the treatment without AMF was due to the control treatment without AMF getting a higher dose of fertilizer, especially urea fertilizer, which was 0.5 gram/polybag while the plants given AMF only got 0.25 gram/polybag.Leaf chlorophyll of Clitoria ternatea from different AMF inoculation in various saline soil types can be seen in Table 6.

Leaf proline level
Based on the results of the analysis of further tests, the leaf proline levels of Clitoria ternatea, the results of different AMF inoculations on saline soils showed significantly different results (P<0.05) with the pattern that AMF EM1 EM2 inoculation was effective in reducing leaf proline levels on moderate saline soils and increasing the dose of salinity significantly (P<0.05)increased the proline content of Clitoria ternatea leaves.Treatment of high saline soils resulted in higher leaf proline levels than medium saline and control soils because the plants experienced salinity stress which resulted in roots being hampered in absorbing water and nutrients.Control treatment without AMF treatment resulted in lower leaf proline levels and significantly different (P<0.05) with AMF treatment.This is because the control treatment was given 20 tons of manure/ha while the treatment given by AMF only got 10 tons/ha.Manure can to reduce salinity stress and improve saline soil characteristics.Leaf proline levels of Clitoria ternatea from different AMF inoculation in various saline soil types can be seen in Table 7.

Table 2 .
AMF colonization of Clitoria ternatea roots in various saline soil types.

Table 3 .
The increase in vertical length of Clitoria ternatea from different AMF inoculations in various soil types.

Table 4 .
The dry weight of the canopy of Clitoria ternatea aged 9 weeks.

Table 5 .
Root dry weight of Clitoria ternatea from different AMF inoculations in various saline soil types.

Table 6 .
Leaf chlorophyll of Clitoria ternatea from different AMF inoculation in various saline soil types.

Table 7 .
Leaf proline levels of Clitoria ternatea from different AMF inoculation in various saline soil types.Note: Different lowercase and uppercase superscripts in the same row and column indicate significantly different (p<0.05).F0: No AMF, 100% fertilizer dose.FU: Superior AMF / comparison, 50% fertilizer dose.EM1: FMA IPB 1, 50% dose of fertilizer.EM2: FMA IPB 2, 50% dose of fertilizer.EM3: FMA IPB 3, 50% dose of fertilizer.T0: Control / non saline soil.T1: Medium saline soil.T2: High saline soil.Saline soil increases the proline content of Clitoria ternatea leaves.The high saline soil reduced the amount of leaf chlorophyll.AMF could help Clitoria ternatea plants cope with saline stress and reduce fertilizer use by 50% of the dose.The addition of AMF increased the total vertical length gain, canopy dry weight, root colonization percentage and root dry weight.The type of AMF product IPB 2 (EM2) gave the best results.Thank you to the Ministry of Education, Culture, Research and Technology of the Republic of Indonesia, Directorate General of Higher Education, Research and Technology for providing funding for the National Competitive Applied research.