Direct seeding of Ceiba pentandra and Leucaena leucocephala using seed briquette with AMF and DSE inoculations to support a green economy

This research is an alternative technology to support a green economy for environmental sustainability. This paper was performed to reveal the relation between seed briquettes inoculated with the fungi of arbuscular mycorrhiza (AMF) and dark septate endophytes (DSE) in direct seeding of C. pentandra and L. leucocephala seedlings on marginal land in Parungpanjang, Bogor, Indonesia. A randomized block design was used in this research with three blocks and found that seed briquettes with AMF and DSE inoculated have significantly influenced the survival of seedlings of both species 12 months after planting. Direct sowing with seed briquette inoculated with AMF (48%) produced the highest seedling survival in C. pentandra, followed by seed briquette injected with Dendrothyrium sp. CPP1.1.4 (44.5%) in L. leucocephala. In C. pentandra and L. leucocephala, seed briquettes increased seedling survival compared to direct sowing without briquettes. Seedling growth was considerably impacted by AMF and DSE inoculations in L. leucocephala but not in C. pentandra. AMF and DSE can improve the height and diameter of direct-seeded L. leucocephala seedlings. Our research found that using seed briquettes and applying DSE has a high potential for increasing the success of direct seeding in assisting various reforestation programs in Indonesia.


Introduction
Direct seeding is a well-known reforestation technology that has numerous advantages, including requiring less work and expense and being able to be applied to large-scale programs [1,2].Increasing the success of direct sowing can use several methods, including species selection [3], seed treatment [4], planting location engineering [1], and seed pellets [1, 5], but often failed and lacked hope [6].The failure was generally due to the low seedling survival, low germination percentage, and the delay of seedling growth [7], as well as increased seed predators, weeds, intense sunshine, severe temperatures, and drought stress conditions [8].
Seedball/seed pelleting has been widely used to rehabilitate degraded land with encouraging results [1,5].Previously, research on direct seeding using seed balls had been undertaken [9], but this study employed a very tiny size (weight 1 g), making it less capable of providing optimal seed germination and growth, particularly on marginal land [5].One of the seedball types, seed briquettes, has been shown to improve seedling survival and growth.

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To promote seedling survival and growth, biofertilizers such as AMF and DSE can be inoculated into the briquette.Mycorrhiza promotes seed germination by boosting phytohormone production and enzyme activity [10].Meanwhile, DSE has colonized potential plant roots both intercellularly and intracellularly without generating disease, promoting seed germination [11].
The use of seed briquettes inoculated with the fungi of arbuscular mycorrhiza (AMF) and DSE to enhance seedling survival and growth must be investigated.Therefore, this study aimed to evaluate the effectiveness of seed briquettes inoculated with AMF and DSE in direct seeding of C. pentandra and L. leucocephala seedlings on marginal land in Parungpanjang, Bogor, Indonesia.C. pentandra has been reported to have a wide ability to grow in various lands such as dry land, yards, and rice field bunds [12] while L. leucocephala, can fix nitrogen from the atmosphere [13] and have great potential for application in direct seeding [5].Hopefully, in the future, seed briquette technology inoculated with AMF and DSE in direct seeding can support the realization of a green economy with environmental sustainability.

Time and location research
C. pentandra seeds were collected from Parungpanjang special purpose forest area (KHDTK), Bogor, West Java (6°23'00.2"S 106°31'07.6"E), while the L. leucocephala seeds were collected from Jatinangor, Sumedang, West Java (6°53'23.9"S 107°45'27.9"E).This research was taken almost ten months from February to December 2021.The preparation for the AMF was carried out at the Laboratory of Microbiology, PT.Intidaya Agro Lestari, Bogor while the DSE isolates were prepared at the Indonesian Soil Research Institute, Bogor (Laboratory of Soil Microbiology).
2.3.Research procedure 2.3.1.Seed briquette.Seed briquettes were made manually using a soil composition (10%), lime (10%), compost (40%), charcoal (30%), and tapioca (5%).The materials were mixed evenly and then tapioca as glue that had been thawed with hot water to form a batter ready for molding.The dough is then placed into melts with the size of 5 cm in diameter and 3 cm in thickness.Seed briquettes are dried for 2-3 days to prevent the growth of fungi and premature germination during storage.Five seeds were put into a briquette then all parts of the seed were wrapped in briquettes.The weight of briquettes containing seeds was about 45-50 g.The seed briquettes were made from organic materials mixed with lime so they have a high nutrient content, such as C organic 4.69%, Ca 33.30 Chol (+) kg -1 , N total 0.56%, K 4.83 Cmol (+) kg -1 , Na 6.36 Cmol (+) kg -1 , P 2 O 5 2080.5 ppm, Mg 9.41 Cmol (+) kg -1 , C/N ratio 8.37, CEC 10.68 Cmol (+) .

DSE and AMF preparations.
For DSE-based fertilizer, three isolates of DSE fungi, i.e., CPP 1.1.4(Dendrothyrium sp.), KSP1 (Cladosporium sp.), and MM 15 (an unidentified isolate), were cultivated in a substrate composed of bran, sawdust, and coarse corn flour.The arrangement of 1000 g of DSE carrier material required 200 g bran, 600 g sawdust, and 200 g coarse corn flour.The materials were thoroughly mingled and stirred with distilled water until the moisture content reached approximately 60%, packaged in autoclave-resistant plastic, and then placed in an autoclave.After the first autoclave process is complete, leave it for 24 hours, then the material is autoclaved again and cooled.DSE fungal inoculum, namely CPP 1.1.4,KSP1, and MM 15, at a rate of 1% of the weight or volume of the carrier material, is inoculated on the carrier material, then stored at room temperature so that DSE can grow and develop on the carrier material so that it is ready to be used in this research.

Experimental design and data analysis
Two tested species were set in separate experimental designs in the field at KHDTK Parungpanjang, Bogor.Testing the effectiveness of biofertilizers (AMF and DSE) used a randomized block design with three (3) blocks.The trial was established by using 10 biofertilizers treatments, i.e.: Seed (control) (a); seed with AMF inoculation (b); seed with CPP 1.1.4(c); seed with DSE isolate MM 15 (d); Seed with KSP.1 (e); seed briquette (f); seed briquette/SB with AMF inoculation (g); SB with CPP 1.1.4(h); SB with MM 15 (i); SB with KSP 1 (j).One block in each treatment consists of 10 planting plots and each sowing plot was sown with 50 seeds or seed briquettes.
The data were analyzed separately on each species based on randomized block design to test the effect of the seed briquette and biofertilizers for seedling survival, seedling height, and root collar diameter using ANOVA.To compare significant differences in the means, DMRT (Duncan's Multiple Range Test) at a significance level of p<0.05.The SAS 9.1 for Windows was used for statistical analysis.

Results and discussion
The survival of C. pentandra and L. leucocephala seedlings 12 months after planting with inoculation using both AMF and DSE had a significant effect (Figure 1).The best seedling survival of C. pentandra was obtained by seed briquette inoculated with AMF using direct seeding (48%) (ST-7), although this treatment was not significantly different from treatments ST-8 and ST-10 (Figure 1a).Meanwhile, the highest seedling survival in L. leucocephala was obtained from direct seeding using a seed briquette inoculated using CPP 1.1.4(44.5%) (ST-8); although not significantly different from treatment ST-6, ST-7, ST-9, and ST-10, but significantly different from ST-1 and ST-2 (Figure 1b).
In general, seed briquettes have higher seedling survival rates compared to using only seeds in direct seeding in the field trial (Figure 1).A previous study found that seed briquettes can improve the speed and germination percentage [1] and the effect was like seed priming [14].Seed briquettes also increase protection from biotic and abiotic stresses, biological control capacity, supply of growth regulation nutrients, moisture attraction, and influence of the micro-environment [5].These results indicate that direct seeding with seed briquettes can provide the best conditions for germination of C. pentandra and L. leucocephala in direct planting in the field on marginal land such as the soil at KHDTK Parungpanjang, Bogor, West Java, which has characteristics of low C-organic content, CN ratio, and base saturation, in addition to moderate N, P, and CEC content and acidic pH.
Two planting seeds methods (seeds planted directly in the field and direct seeding with seed briquette) also affect the effectiveness of the two isolates used in this research.The role of DSE and AMF in supporting seedling survival can be seen from the colonization of AMF and DSE in C. pentandra and L. leucocephala seedlings grown by direct seeding (Figure 2).While both plant species have been reported to have symbiosis with AMF, this research is the first report that DSE can establish symbiosis with C. pentandra and L. leucophala.The possibility of native AMF and DSE having infected the roots of the two tested species has also been shown by the presence of colonization of the uninoculated seed briquettes (Figure 2).The condition where natural native AMF infected a species was also found in-the-field implantation test study [15], however, colonization of trees inoculated with AMF and DSE was higher, so this indicated that the two fungal inoculants were actively symbiotic with both tree species.The high percentage of colonization on C. pentandra seedlings however was not related to a seedling survival level, although for some treatments, inoculation of isolate MM-15 on seeds and seed briquettes of L. leucocephala gave the highest seedling survival.The role of DSE and AMF in promoting various plant growth has been reported in several studies [11].However, the use of DSE especially to escalate the growing survival of L. leucocephala and C. pentandra seedlings in with poor soil conditions (marginal lands) has not been widely carried out, furthermore, there is not much information about the physiological effects of DSE and AMF on the germination of second seeds of these tree species in the field exposed to different types of biotic and abiotic stresses.The strigolactones function in plants as signaling molecules at the onset of AMF colonization.They induce fungal metabolism and branching, simultaneously initiating seed germination.[16].DSE also has a role in seed germination and influences growth survival in the field, especially on L. leucochepala on seed briquettes in this study.This capability is likely a vital stage in their formation within the host plant and their subsequent facilitation of seed germination, and so are the DSE isolate species used in this study have been reported to enhance the growth of their host plants in different  AMF and DSE inoculations significantly affected the growth of L. leucocephala seedlings but did not significantly impact the growth of C. pentandra seedlings (Table 1).In L. leucocephala seedlings, inoculation of CPP 1.1.4(Dendrothyrium sp.) on the seed briquettes gave the best seedling height and diameter, i.e., 38.2 cm and 4.1 mm, respectively (Table 1).These results, especially for L. leucocephala seedling height, were not significantly different from seedlings inoculated with MM-15 (unidentified isolate) and KSP 1 (Cladosporium sp.).The MM-15 and KSP 1 inoculations also provided L. leucocephala seedling height growth on direct seeding using seeds that were not significantly different from the seed briquettes inoculated with the two DSE (Table 1).
The existence of AMF and DSE in the roots of C. pentandra and L. leucocephala seedlings also influenced the seedlings' growth after planting in the field.In general, AMF and DSE inoculation successfully boosted the diameter and height of L. leucocephala seedlings grown from direct seeding.AMF assists plants in augmenting their ability to absorb nutrients and developing resistance to drought and various abiotic stresses [18] in the experimental fields used in the study with nutrient-poor and acid soil conditions, as well as DSE which has the same effect on the two tree species.The presence of AMF and DSE seems to be associated with increased auxin and cytokinin production which promote plant growth as well as other secondary metabolite products produced by the two types of fungi [19].Pretreatment of young seedlings by using efficient AMF and DSE not only makes them stronger but also helps in their establishment after out-planting in the field [20].In this research, the AMF used was a consortium of several species, i.e., Glomus sp.1, Glomus sp.2, Gigaspora sp., and Acaulospora sp.Consortium of AMF species had better colonization and growth of host plants than the application of individual AMF species [21].AMF symbiosis with young plants can improve plant protection against oxidative damage caused by water stress, plant nutrition, and tolerance to abiotic stress [22] generally AMF inoculants in Indonesia are a consortium of several types of AMF, while DSE is still rarely used for field research in Indonesia either in the form of single strains or consortium.In the context of DSE, multiple instances can function as influencers of plant growth by protecting plants against biotic and abiotic stress [23].C, N, and P nutrients are essential nutrients, especially at the beginning of their growth.The role of DSE as a protector against pathogens (biotic disturbances) and abiotic stress has led to speculations that DSE can be a biological agent in protecting plants and promoting efficient use of water.Further studies are needed to prove this with field observations for several years in the short and long term.However, we have proven in field tests that in addition to AMF, DSE can stimulate the growth of C. pentandra and L. leucocephala so that they can adapt to marginal land with low fertility.Therefore, the use of DSE symbiotic technology with these two tree species to rehabilitate degraded lands or marginal lands has the potential to be carried out on a wider scale in a sustainable manner.

Conclusions
AMF and DSE inoculations significantly affected the endurance of L. leucocephala and C. pentandra seedlings within 12 months after sowing.The best survival of C. pentandra seedlings was obtained by direct seeding using AMF-inoculated seed briquettes (48%), while seed briquettes inoculated with Dendrothyrium sp. had the best result on L. leucocephala (44.5%).Seed briquettes provided higher seedling survival compared to direct seeding using only seeds for both C. pentandra and L. leucocephala.AMF and DSE inoculations significantly affected the growth of L. leucocephala seedlings but not C. pentandra seedlings.The direct seeding of L. leucocephala seedlings saw an augmentation in height and diameter through the inoculation of AMF and DSE.The best seedling performance of L. leucocephala was found in seed briquettes inoculated with Dendrothyrium sp., indicated for seedling survival (30.6%), height (38.2 cm), and diameter (4.1 mm).Our findings indicate that there is substantial potential to enhance the efficiency of direct seeding by using seed briquettes and DSE inoculation.
Therefore, further development is warranted to optimize their application in the field.The expectation is that the results of this research can be applied as an alternative to a green economy in supporting efforts for environmental sustainability.

Figure 2 .
Figure 2. Percentage of colonization of AMF and DSE on the direct seeding of C. pentandra and L. leucocephala.Notes: see Figure 1 for seed treatment information.
-2 ST-3 ST-4 ST-5 ST-6 ST-7 ST-8 ST-9 ST-10 Percentage of colonization (%) Seed treatment on Ceiba pentandra -2 ST-3 ST-4 ST-5 ST-6 ST-7 ST-8 ST-9 ST-10 Percentage of colonization (%) Seed treatment on Leucaena leucocephala b conditions a biotic and abiotic pressure conditions (see section 2.1).Nevertheless, the role of mycorrhizae and DSE in seed germination especially for its physiological effect [17].The structure of AMF and DSE from L. leucocephala seedlings roots is shown in Figure 3.The difference in the structure of the colonies on the roots of the L. leucocephala seedlings indicates the presence of DSE and AMF.The attendance of DSE in roots was indicated by the formation of microsclerotia (Figure 3. A1, and A2).The attendance of AMF in the roots indicated the presence of vesicles (Figure 3. B1 and B2).
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Table 1 .
Seedling height and diameter of C. pentandra and L. leucocephala at 12 months after sowing.
**= significantly confident at the 99% level, *= significant at the 95% confidence level, ns= not significant at the 95% confidence level.Different letters (a, b, c, ab, and bc) in the same column indicate significant differences at P ≤ 0.05 between treatments.