Suppression on plant-parasitic nematodes using a soil fumigation strategy based on ammonium bicarbonate and its effects on the nematode community

Banana production is severely hindered by plant-parasitic nematodes in acidic, sandy soil. This study investigated the possibility of applying a novel fumigation agent based on ammonium bicarbonate as a strategy for controlling plant-parasitic nematodes under sealed conditions. Moreover, its effects on the nematode community in pot and field experiments were also measured using morphology and feeding-habit based classification and the PCR-DGGE method. Results showed that a mixture (LAB) of lime (L) and ammonium bicarbonate (AB) in suitable additive amounts (0.857 g kg−1 of L and 0.428 g kg−1 of AB) showed stronger nematicidal ability than did the use of AB alone or the use of ammonium hydroxide (AH) and calcium cyanamide (CC) with an equal nitrogen amount. The nematode community was altered by the different fumigants, and LAB showed an excellent plant-parasitic nematicidal ability, especially for Meloidogyne and Rotylenchulus, as revealed by morphology and feeding-habit based classification, and for Meloidogyne, as revealed by the PCR-DGGE method. Fungivores and omnivore-predators were more sensitive to the direct effects of the chemicals than bacterivores. This study explored a novel fumigation agent for controlling plant-parasitic nematodes based on LAB and provides a potential strategy to ensure the worldwide development of the banana industry.


Results
Nematicidal effect of LAB. The effects of different concentrations of L, AB and LAB on nematode numbers are shown in Fig. 1a. For all the results, the maximum population was found in the CK (pot soils with no additives), AB1 and L4. The total number of nematodes showed a continuous decrease in the AB and LAB treatments and an irregular fluctuation in the L treatment. The minimum value was observed after treatment with AB and LAB in the highest amount added, and less than 300 individuals per 100 g d.w. soil were identified when the amount added was more than 0.857 g of L and 0.428 g of AB. Thus, this concentration (LAB4) was selected for further study. Compared with the AB treatment, the LAB treatment improved the effects for reducing the nematodes numbers by 51.4%, 70.4%, 45.0%, 55.7% and 83.7% with the increase of additional concentrations, respectively.
For treatments with different soil moisture contents and the control, the total number of nematodes followed the same trend, gradually decreasing with increasing soil moisture content. Treatment with LAB significantly reduced the nematode number compared with the control when the soil moisture content was 10%, while no significant difference was observed when the moisture was higher than 30%, which is unsuitable for survival (Fig. 1b). For treatments with different temperatures compared with the control, the number of nematodes in the soil gradually decreased with increasing temperature values. The nematode number in the soil of the LAB treatment was significantly lower than that of the control when the temperature was less than 40 °C (Fig. 1c).

Physicochemical properties of soil samples from LAB treatments at different concentrations.
Compared with the previously published results for different nematicidal agents, the utilization of LAB more efficiently killed the nematodes, representing the most efficient strategy. Therefore, the Scientific RepoRts | 5:17597 | DOI: 10.1038/srep17597 physicochemical properties of soil samples treated with different concentrations of LAB and the control (CK) with no additives were determined. As shown in Table 1, the pH value overall continuously increased with increasing amounts of LAB and higher than that in CK; the NH 4 + -N content increased to 28.99 mg kg −1 in the 2 treatment (0.428 g of Lime and 0.214 g of ammonium bicarbonate), slowly decreased to 11.70 mg kg −1 when 2.568 g of lime and 1.284 g of ammonium bicarbonate were added, and then suddenly increased to the highest value of 136.23 mg kg −1 in the treatment with the highest addition amount. The NO 3 − -N content continuously increased with increasing addition of LAB when the amount added was less than 3.424 g of lime and 1.712 g of ammonium bicarbonate. After this, the NO 3 − -N content suddenly decreased to the lowest value of 29.64 mg kg −1 . The total nitrogen content of the soil samples increased with increasing LAB, ranging from 0.36 mg kg −1 in the a treatment to 0.51 mg kg −1 when the highest amount was added, representing the accumulation of nitrogen in the soils with added LAB.
Nematicidal effect of different ammonia production materials in Petri dishes. The nematicidal effect of aqueous solutions of LAB, AB, CC and AH evaluated in a Petri dish experiment are shown in Fig. 2. Both diluents of LAB almost completely killed the nematodes. In the 20-fold diluent experiment, compared with the control (Petri dish with no additives) with mortality of 12%, the corrected mortality of the nematodes for LAB, AB, CC and AH treatments were 98%, 95%, 54% and 93%, respectively. In the 50-fold diluent experiment, the values were 98%, 85%, 36% and 86%, respectively. These Petri dish experiment results indicate that LAB is the most efficient nematicidal agent.
Nematicidal effect and nematode community analysis after fumigation in the pot experiment by morphology. For the total number of nematodes after fumigation, as shown in Table 2, no significant difference was observed in the nematicidal effects between AB and AH treatments. Compared with these two treatments and L control, the application of LAB significantly reduced the nematode  number, indicating that LAB is the most efficient soil pretreatment agent. To determine the effects of different ammonia production materials on the nematode distribution, soil samples from the pot experiment collected after fumigation were selected for nematode taxa analysis. As shown in Table 2, all trophic groups were significantly affected by fumigation. No significant difference in the number of bacterivores was observed among the three treatments and L control, while the values were significantly lower than that in CK. The LAB and AH treatments significantly decreased the fungivores number compared to the AB treatment and CK and decreased the omnivores-predators number compared with L and CK. Both LAB and AB killed more plant parasites than the AH treatment and CK. A total of 17 taxa were counted and identified in the soil samples collected after fumigation (Table 2). Bacterivores, with 7 taxa, were the most diverse trophic group. No significant difference of the Acrobeloides number was observed in the AB, LAB, AH treatments and L control, and the values were significantly lower than that in the CK. The AB, LAB and AH treatments significantly decreased the Chiloplacus number compared to CK. Moreover, the LAB and AH treatments and L control significantly decreased the Cephalobus number compared with the CK and AB treatment, while LAB and CK showed no significant difference in Mesorhabditis number, which significantly increased in the L control and AH treatment. No significant difference in the number of Prismatolaimus, Acrobeles and Monhystera were observed among the treatments and controls. Four species of plant parasites were identified in the soil samples: Rotylenchulus, Meloidogyne, Pratylenchus and Tylenchus. The LAB and AH treatments showed the best fumigation effect for Rotylenchulus compared to CK, L and AB treatment. The numbers of Meloidogyne in the AB and LAB treatments were significantly lower than in other treatments and the two controls. Significantly lower numbers of Tylenchus were observed in the L, AB and LAB treatments, followed by the AH treatment, while the values were highest in the CK. All treatments and controls showed no significant difference in Pratylenchus number. The number of Tylencholaimus, Aphelenchus and Aphelenchoides, belonging to fungivores, and Mylonchulus, Oxydirus and Aporcelaimus, belonging to omnivores-predators, differed for all treatments and controls.
Nematicidal effect and nematode faunal analysis at the end of the pot experiment by morphology. At the end of the pot experiment, significant differences in the numbers of plant parasites and total nematodes were found among the three treatments and two controls for both the banana roots and the soil, and the lowest values were observed in the LAB treatment, followed by the AB treatment, AH treatment, L control and CK (pot soils with no additives) for roots (Fig. 3) and by the AB treatment, L control, AH treatment and CK for soil ( Table 2).
For the tropic groups in the soil, LAB treatment showed the highest number of bacterivores. No significant difference in the fungivores number was observed among the treatments, and the values were significantly lower than those in the CK. Significant difference in the numbers of plant parasites was observed among the three treatments and the two controls. The LAB treatment showed the lowest number, followed by the AB treatment, L control, AH treatment and CK, indicating that LAB is the best fumigation agent. The highest number of omnivores-predators was observed in the L, LAB treatments and CK, followed by the AB and AH treatment ( Table 2).
A total of 15 taxa were counted and identified in the soil samples collected at the end of the pot experiment ( Table 2). The LAB treatment showed the highest number of all 6 taxa belonging to bacterivores: Mesorhabditis, Acrobeloides, Cephalobus, Prismatolaimus, Chiloplacus and Acrobeles, with the first three significantly higher in number compared with the other treatments and with the controls.

Omnivores-Predators
Mylonchulus  Nematode community analysis after fumigation in the pot experiment using PCR-DGGE. The DGGE fingerprint analyses of the soil samples after fumigation are shown in Fig. 4. Based on a UPGMA method analysis, the DGGE lanes grouped into 3 clusters were distinctive. The three-replicate cluster for the soil treated by LAB was similar to that from the AB treatment and different from the other clusters (Fig. 4). The strengthened bands in the DGGE gels were excised for sequencing, and 15 bands were successfully sequenced and analyzed by a BLAST search. The highest identities based on comparison to known and putative species in the NCBI database are shown in Table 3, and all the identified bands belonged to plant-parasites and bacterial-feeders. For the plant-parasites, the weakened bands (N14 and N15) detected in the LAB and AB samples were identified as Meloidogyne javanica and Meloidogyne incognita. The N5 and N11 bands observed in all the samples were identified as Tylenchus arcuatus isolate wb8 and Helicotylenchus dihystera and were weakly strengthened in the LAB and AB treatments. No obvious differences in intensity were observed for bands N7, N8 or N12, identified as Ditylenchus brevicauda, Rotylenchulus reniformis and Rotylenchulus reniformis, respectively. For the bacterial-feeders, no obvious differences in the intensities of the bands were detected, except for in bands 1 and 2, identified as Prismatolaimus intermedius and Cephalobus cubaensis, respectively, which showed decreased intensities in the LAB and AB treatments.
Nematode community analysis at the end of the pot experiment using PCR-DGGE. The DGGE fingerprint analyses of the soil samples collected at the end of the pot experiment are shown in Fig. 5. Based on a UPGMA method analysis, the DGGE lanes grouped into 2 clusters were distinctive. The three-replicate cluster for the soil amended with LAB was distinct from that of the other samples.
The strengthened bands in the DGGE gels were excised for sequencing, and 15 bands were successfully sequenced and analyzed by a BLAST search (Table 4)  bands in the treatments of AB and LAB and in the L control were weaker than in the AH treatment and in CK.

Discussion
This study, to our knowledge, marks the first investigation of a novel fumigation agent to be applied to soil as a strategy for controlling plant-parasitic nematodes in acidic, sandy soil which was monocultured banana for more than ten years. Because ammonium bicarbonate under alkaline conditions is more readily volatilized into ammonia 32 and the nematicidal activity of ammonia and ammonia-releasing organic amendments is generally stronger in alkaline soils 33 . Both ammonium bicarbonate alone and ammonium bicarbonate with lime (two-fold dose relative to ammonium bicarbonate) were investigated as fumigation agents in nematode-affected soil in Hainan province, China, of which the pH value generally reported  Table 3. Closest relative for the DGGE bands with the greatest similarities in BLAST database search after fumigation from the pot experiment. is less than 7 13 . Different concentrations of ammonium bicarbonate alone and ammonium bicarbonate with lime were tested, revealing that higher additive amounts produced enhanced ability for suppressing nematodes population. The result was supported by earlier findings that ammonium bicarbonate was reported to suppress Belonolaimus longicaudatus, one kind of plant-parasitic nematode, in the field 34 .
In addition, the mixture of ammonium bicarbonate and lime possessed significantly higher suppressing effects compared to the same dose of ammonium bicarbonate alone, most likely due to the higher pH value generated by the addition of lime 32 . The soil pH is consequently the crucial driving factor in the production of ammonia 32 . In acid soils, ammonium-nitrogen (NH 4 + -N) predominates, while at higher pH, NH 3 would be more prevalent 35 . When the concentrations of the mixture were more than that in LAB4, pH was greater than 7, also showing a better nematicidal ability. The application of excess ammonia-releasing and ammonium compounds is not only phytotoxic and costly; it may also contaminate ground water through the leaching of nitrate, which is produced from ammonia by the activity of soil microorganisms 23 . Our results showed that the nematode number was less than 300 individuals per 100 g d.w. soil when the amount of the mixture added was more than 0.857 g kg −1 of L and 0.428 g kg −1 of AB, indicating a suitable dose to be selected for further study. In this experiment, an increasing total nitrogen content with increasing additive amounts were also observed, while the NH4 + -N content increase did not correspond with the additive amount due to the soil ammoxidation ability 23 . In addition, LAB was further confirmed to be the most effective fumigation agent when compared to the suppressing effect of AB, calcium cyanamide which was previously reported to be an effective fumigation agent 36 , and ammonium hydroxide which was also observed to be the most nematicidal compound compared with ten ammonia-releasing compounds through pot experiments 23 . The superior effect of LAB compared with ammonium hydroxide may be because the latter one is not suitable where the soil texture makes sealing of the tine furrow difficult; for example, on very heavy or sandy soils 37 . The density of nematodes was reduced in wet sites. The soil under the low oxygen condition produced the anaerobic metabolism product which can suppress the number of nematodes 38,39 . In our study, the mortality is so high when the soil moisture content was greater than 50%.
During one season of pot experiments, the nematode community structure was investigated by morphology and feeding-habit based classification because nematodes have high species diversity and belong to different feeding groups, including bacterivores, fungivores, plant parasites and omnivores-predators 40 , and are ubiquitous members of the soil faunal community in terrestrial ecosystems 41 . It is particularly important and significant to investigate the effects of fumigation on the plant-parasitic nematode population and community structure. Plant-parasitic nematodes which are among the most difficult crop pests to control may reduce crop yield through direct cell destruction and the vectoring of viruses or indirectly by facilitating the invasion of fungi and bacteria through their feeding and movements through roots 42 . Our results showed that, LAB was an excellent fumigation agent and showed a better suppression ability for Meloidogyne than AH treatment and the two controls after fumigation and harvest, and for Rotylenchulus than all treatments and the two controls after harvest. No significant difference in the values of Meloidogyne number in LAB and AB treatments was observed after fumigation and harvest, nevertheless, the values in the LAB treatment were both lower. Meloidogyne is the major nematode pest  affecting banana (Musa spp.) production as well as a wide range of other agricultural crops across the globe 11,43 . Rotylenchulus reniformis can suppress crop yield of 60-74% 44 . Similar observations that soil treated with different fumigants, such as methyl iodide, 1, 3-dichloropropene, or propargyl bromide, successfully suppress plant-parasitic nematodes, especially Mesocriconema and Meloidogyne, have previously been reported 16 . However, many of these alternative soil fumigants are devastating to soil ecosystems 21 .
The alternative fumigant described in the present study, which has previously been used as a general inexpensive fertilizer, may be widely pursued. In addition, our nematode community structure results also indicated that the abundance of fungivores and omnivores-predators was strongly reduced by all fumigants, most likely due to direct fumigant toxicity and alterations to the fungal decomposition channel 45 , while the abundance of bacterivores did not differ extensively among the treatments. In agreement with our results, previous studies have also shown that fungivores were more sensitive to the direct effects of chemicals than were bacterial-feeders 45 . However, morphology and feeding habit-based classification exhibits serious limitations in terms of taxonomic identification and low sample throughput 46 . Only experts with the knowledge and skills to identify nematodes can conduct such analyses on a regular basis, which poses a major obstacle in the analysis of nematode communities 47 . Thus, PCR and sequence-dependent molecular biological methods, including denaturing gradient gel electrophoresis (DGGE), were applied to further study the nematode communities. The nematode primers M18F and M18R from Bhadury and Austen 48 were used to fingerprint nematode diversity. After fumigation and at the end of the pot experiment, as revealed by PCR-DGGE, LAB application increased the nematode community structure (i.e., more bands); Based on an UPGMA method analysis of the DGGE lanes, the LAB treatments clustering with the AB treatment were distinctive to AH, CK and the L control after fumigation and then AH and CK at the end of the pot experiment, indicating that LAB application significantly altered the nematode community.
Most of the bands excised from the gels demonstrated high sequence similarity with nematodes, showing that the modified primer and the DGGE target resolved the nematode 18S rDNA signal in these soils 49 . The nematode species known to cause the most serious damage to bananas are those belonging to the migratory endoparasites, including the sedentary parasite Meloidogyne spp. 10 . In the present study, the results of morphology and feeding habit-based classification and the DGGE both revealed that the LAB treatment resulted in effectively suppressing ability for plant-parasitic population. Although the presence of bands representing different nematode taxa were relatively weaker than those observed by morphology and feeding-habit based classification, elucidating the factors that determine the abundance and composition of soil nematode communities is still a promising approach when investigating and understanding the effects of different fumigants in this study.
According to reports, ammonia is the source of more than 95% of the chemical nitrogen fertilizer currently produced in the world, and may be used directly as a fertilizer or converted to ammonium salts, nitrates, or urea 50 . It is almost half a century since ammonium bicarbonate was first introduced in European countries as a nitrogen fertilizer 51 . In addition, in acid soils, a marked decrease in acidity should occur in the soils which are amended with free lime to adjust the soil pH 52 . Single application of lime maintained increased soil pH and crop yield for 16 to 27 years 53 . Sun et al. 52 also found ammonium bicarbonate can provide a potential strategy for ensuring healthy cucumber, watermelon and melon crops. Thus, the combined results showed that the utilization of LAB as a fumigant can effectively suppress soil nematode damage in acidic, sandy soils, especially those affected by plant-parasitic nematodes and provides a potential strategy to ensure the worldwide development of the banana industry.

Methods
Source of soil. Soil was collected at the "Wan Zhong" orchard (18°23′ N, 108°44′ E), Le Dong County, Hainan Province, China from areas monocultured with banana for more than ten years. The area has a tropical monsoon climate with approximately 600-3000 individuals of nematodes per 100 g d.w. soil. The soil was sandy and had an organic matter content of 7.6 g/kg, an available potassium content of 278.2 mg/ kg, an available phosphorus content of 173.6 mg/kg, a pH value of 5.6, a total nitrogen content of 0.4 g/ kg, an NH 4 + -N content of 10.3 mg/kg, and a NO 3 − -N content of 143.9 mg/kg.

Selection of the best additive amount of ammonium bicarbonate as a fumigant to control nematodes.
To select the best additive amount of the ammonia production material, ammonium bicarbonate, as a fumigant to control nematodes, eight concentrations, including 0.107 g, 0.214 g, 0.321 g, 0.428 g, 0.856 g, 1.284 g, 1.712 g and 2.568 g, were applied to pot soils (1 kg d.w. each) without banana planting and termed AB1, AB2, AB3, AB4, AB5, AB6, AB7 and AB8, respectively, were established. Because ammonium bicarbonate under alkaline conditions more readily produces volatilized ammonia, the pot soils containing ammonium bicarbonate mixed with a 2-fold concentration of lime termed LAB1, LAB2, LAB3, LAB4, LAB5, LAB6, LAB7 and LAB8, respectively, were also established. The soils in the subsequent treatments were used to compare the nematicidal effects for equivalent concentrations of ammonium bicarbonate. Pot soils with no additives, termed CK, and just containing lime, termed L1, L2, L3, L4, L5, L6, L7 and L8 were used as controls. Each treatment (16 in total) or control (9 in total) was composed of three blocks, with each block containing three pots. Each pot surface was sealed using a plastic film, leaving four ventilation holes at the bottom. The soil was mixed thoroughly with the moisture content adjusted to 20% by adding distilled water and incubated at room temperature for 15 days. After that, three subsamples combined to form a composite sample (approximately 200 g) were collected from each block for calculating the total number of nematodes. The effects of the moisture content values (adjusted to 10%, 30%, 50% and 100%) and the temperature (adjusted to 20, 30, 40 and 50 °C) on the nematicidal ability of the mixtures of ammonium bicarbonate and lime (LAB) with the best additive concentration were examined. Pot soils without any amendment were used as controls. Treatments (8 in total) and controls (8 in total) were set as previous paragraph described. The total number of nematodes was calculated also as previous paragraph described.
Soil chemical analysis. The pH was determined using a glass electrode meter in a suspension of 1:5 (w/v) soil: water. The total soil nitrogen content was determined by Kjeldahl digestion followed by

Comparison of the nematicidal effect of different ammonia production materials in Petri dishes.
For the Petri dish experiment, ammonia production materials for all the treatments containing the same content of N, termed LAB (0.857 g of lime and 0.428 g of AB, d.w.), AB (0.428 g of AB, d.w.), CC (0.257 g of calcium cyanamide, d.w.) or AH (0.139 g of ammonium hydroxide, d.w.) were dissolved in 10 ml of deionized water, then 10 ml of a 20-or 50-fold dilution of each material was transferred to Petri dishes plated with 150 individual live nematodes containing the randomly nematode community with different genera collected from soil which has been planted banana more than ten years. An equal volume of deionized water was used as a control. The corrected mortality of nematodes was calculated after standing for 24 h. Nematodes were considered alive if they moved or appeared as a winding shape 54 and were considered dead if they did not move when probed with a fine needle 55 . Then, the nematodes in each treatment were transferred to distilled water for 48 h to ascertain whether dead nematodes regained mobility or not. The corrected nematode mortality percent was calculated according to Abbott's Formula: The total number of nematodes was calculated after sealing and incubating at room temperature for 15 days. The total nematodes per 100 g d.w. soil were collected for subsequent DNA extraction prior to PCR-DGGE analysis. After fumigating the soil, the pot soils were supplemented with cattle manure compost (30 g kg −1 soil, d.w.) and transplanted with banana seedlings (Musa AAA Cavendish cv. Brazil) provided by Hainan Wan Zhong Co., Ltd., China. The germ-free banana seedlings were first grown in nursery cups for 30 days and then transplanted into pots after growing 5-6 true leaves. After planting for 80 days, three plants were randomly chosen from each treatment, and the roots were uprooted from the pots and shaken gently to collect the soil samples and washed to remove the soils for the root samples. The plant parasites were collected from root. Three soil subsamples combined to form a composite sample were collected from each block, triplicate composite samples were prepared to calculate the total number of nematodes, and the total nematodes per 100 g d.w. soil were collected for subsequent DNA extraction.
Nematode community analysis. The nematodes in the roots were extracted by using a beater to break the roots and then collected by a modified Baermann funnel methodology 56 . The nematodes in the soil were extracted using sieving and the modified Baermann funnel methodology 56 , and the nematode populations were expressed per 100 g d.w. soil. Briefly, 100 g of soil was suspended in 1.5 L of water and sieved sequentially through 0.096 mm and 0.037 mm sieves. The nematodes were collected through the 0.037 mm sieve and backwashed to 10 ml for counting using a light microscope (OLYMPUS CX41RF) × 40, the nematode taxa classified based on known feeding habitats or stoma and esophageal morphology 57  a combination of three replicates of soil samples collected after fumigation and at the end of the pot experiment were separately analyzed by PCR-DGGE 13 . The nematode 18S rRNA region was amplified using the primers M18F with a GC clamp (5′ -AGRGGTGAAATYCGTGGAC-3′ ) (position 842 to 860 in relation to Caenorhabditis elegans 18S rRNA gene) and M18R (5′ -TCTCGCTCGTTATCGGAAT-3′ ) (position 1,269 to 1,251 in relation to C. elegans) 48 . The PCR mixture and conditions were the same as those described in the original studies 49 . DGGE analyses were conducted using a D-Code Universal Detection System (Bio-Rad Laboratories, Hercules, USA). Twenty μ l of PCR product samples with 5 μ l of loading dye were loaded into the wells of a 6% polyacrylamide gel (acrylamide: bis-acrylamide 37.5: 1) containing a gradient of 30% to 45% denaturants (a 100% denaturant concentration was defined as 7 M urea and 40% v/v deionized formamide). Electrophoresis was performed in 1× TAE buffer at a constant temperature of 60 °C with a constant voltage of 90 V for 10 h. The gel was visualized by silver staining and scanned using a scanner (Epson perfection v33, Seiko Epson Corporation, Japan). Strengthened intensity DGGE bands were excised and sequenced as described by Shen et al. 13 .
The DGGE images were analyzed by the Quantity One software program (Version 4.6.3, Bio-Rad Laboratories) for band detection and intensity measurements. Cluster analysis was performed by the UPGMA algorithm (Quantity One Version 4.6.3, Bio-Rad Laboratories).

Data analysis.
The pot experiments were repeated twice with similar results. Therefore, only the data from one of the pot experiments is presented herein. Analysis of variance (ANOVA) was used to determine differences between the different treatments with respect to nematode abundance. A comparison of means was performed by the Duncan multiple range test with a significance level of P < 0.05 using SAS9.2 (SAS Institute, Cary, N.C.).