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Glufosinate enhances the activity of protoporphyrinogen oxidase inhibitors

Abstract Glufosinate inhibits glutamine synthetase (GS), a key enzyme for amino acid metabolism and photorespiration. Protoporphyrinogen oxidase (PPO) inhibitors block chlorophyll biosynthesis and cause protoporphyrin accumulation, a highly photodynamic intermediate. Both herbicides ultimately lead to plant death by a massive accumulation of reactive oxygen species (ROS) through different mechanisms. We investigated a potential synergistic effect by the mixture of the two herbicide mechanisms of action (MoAs). The tank mix between a low rate of glufosinate (280 g ai ha−1) with an ultra-low dose of saflufenacil (1 g ha−1) provided enhanced herbicidal activity compared with the products applied individually on Palmer amaranth (Amaranthus palmeri S. Watson). The synergism between the two herbicides was also confirmed by isobole analysis and field trials. The herbicide combination provided high levels of efficacy when applied at low temperature and low humidity. Mechanistically, glufosinate caused a transient accumulation of glutamate, the building block for chlorophyll biosynthesis. Consequently, inhibition of both GS and PPO resulted in greater accumulation of protoporphyrin and ROS, forming the physiological basis for the synergism between glufosinate and PPO inhibitors. While the synergy between the two herbicide MoAs provided excellent efficacy on weeds, it caused low injury to PPO-resistant waterhemp [Amaranthus tuberculatus (Moq.) Sauer] and high injury to both glufosinate-resistant and glufosinate-susceptible soybean [Glycine max (L.) Merr.]. Glufosinate enhances the activity of PPO inhibitors through glutamate and protoporphyrin accumulation, leading to increased levels of ROS and lipid peroxidation. The synergism between the two herbicide MoAs can help to overcome environmental effects limiting the efficacy of glufosinate. Future research is needed to optimize the uses for this herbicidal composition across different cropping systems.

Introduction
Glufosinate is sold commercially as a racemic mixture of D-and L-phosphinothricin but only the L-isomer has herbicidal activity (Beriault et al. 1999). Glufosinate-resistant crops (Liberty Link®) are genetically engineered for phosphinothricin acetyltransferase (pat) expression to metabolize L-phosphinothricin into N-acetyl-L-phosphinothricin, a nonphytotoxic compound (Dröge et al. 1992). To date, there have been only five cases of evolved glufosinate resistance in the world: goosegrass [Eleusine indica (L.) Gaertn.] from Malaysia, rigid ryegrass (Lolium rigidum Gaudin) from Greece, perennial ryegrass [Lolium perenne L. ssp. multiflorum (Lam.) Husnot] from New Zealand, L. perenne ssp. multiflorum from Oregon (USA), and L. perenne ssp. multiflorum from California (USA) (Brunharo et al. 2019;Ghanizadeh et al. 2015;Jalaludin et al. 2010;Karn et al. 2018;Travlos et al. 2018). Although glufosinate is an alternative tool to manage multiple herbicide-resistant weeds, it does not translocate well in plants, and weed efficacy is greatly affected by environmental conditions (low light, temperature, and humidity) and plant size (Coetzer et al. 2001;Sellers et al. 2003;Steckel et al. 1997). Glufosinate is a fast-acting and broad-spectrum herbicide targeting glutamine synthetase (GS), a key enzyme for amino acid metabolism and photorespiration in plants (Bayer et al. 1972;Oliveira et al. 2002). GS catalyzes the incorporation of ammonia into glutamate to form glutamine (Bernard and Habash 2009). Glufosinate inhibits GS competing with glutamate for the active site. While GS inhibition leads to ammonia accumulation and changes in amino acid levels, the rapid phytotoxicity results from a massive lightdependent generation of reactive oxygen species (ROS) (Takano et al. , 2020a. Fluxomics refers to small molecule fluxes and networks across different metabolic pathways in systems biology of living cells (Winter and Krömer 2013). Glutamate is an interesting example to study fluxomics, because it occupies a central position in amino acid metabolism. In addition to its role as a substrate for GS, glutamate is also a precursor for proline, arginine, and chlorophyll biosynthesis (Forde and Lea 2007). Protoporphyrinogen oxidase (PPO) is an important enzyme for the chlorophyll pathway, catalyzing the conversion of protoporphyrinogen (protogen) into protoporphyrin (proto) (Lermontova et al. 1997). When PPO is inhibited, protogen leaks out of the chloroplast where the reaction takes place. In the cytoplasm, protogen is converted into proto. Therefore, inhibition of PPO leads to accumulation of proto, the product of the reaction (Lee et al. 1993;Matringe et al. 1989). Herbicides targeting PPO are also fast acting because proto generates ROS in the presence of light, causing lipid peroxidation (Dayan et al. 2019b). Resistance to PPO inhibitors has been reported in 13 species around the world (Heap 2020). In a PPOresistant waterhemp [Amaranthus tuberculatus (Moq.) Sauer] population, a codon deletion (ΔG210) affected herbicide binding to the active site and provided high levels of resistance to lactofen (Dayan et al. 2010;Patzoldt et al. 2006).
Herbicide mixtures are commonly used in agriculture to improve efficacy, increase the spectrum of weed control, and mitigate herbicide resistance (Busi et al. 2019;Zhang et al. 1995). The combination between two herbicides is synergistic when the combined effect is larger than predicted (Sørensen et al. 2007). The fact that glutamate is the precursor for both glutamine and chlorophyll biosynthesis suggests that inhibiting these two pathways at once could lead to catastrophic consequences in plants. We used a fluxomics approach to investigate the fate of glutamate under GS and PPO inhibition. Our hypothesis was that glutamate is diverted to the chlorophyll biosynthesis pathway following GS inhibition. If this is true, glufosinate could enhance the activity of PPO inhibitors by diverting the carbon flow toward proto accumulation in the chlorophyll pathway. Thus, we investigated the interaction between glufosinate and PPO inhibitors, along with the physiological basis for the synergistic effect observed with the herbicide combination.

Plant Growth and Spraying Conditions
Seeds of Palmer amaranth (Amaranthus palmeri S. Watson) were collected in eastern Colorado, and plants were susceptible to both glufosinate and PPO inhibitors. All plant species were grown in 0.3-cm 3 pots filled with soil (Sun Gro Horticulture, Agawam, MA, USA). Greenhouse conditions were 25/21 C day/night, 16-h photoperiod with light intensity of 500 μmol m −2 s −1 and 70% relative humidity (RH). For herbicide applications, a commercial chamber track sprayer (DeVries Manufacturing, Hollandale, MN, USA) equipped with an 8002EVS single even, flat-fan nozzle (TeeJet®, Spraying Systems, Denver, CO, USA) calibrated to deliver 187 L ha −1 spray solution at the level of the plant canopy was used.

Levels of Amino Acids and Glufosinate
Plants of A. palmeri (6-leaf stage) were sprayed with 560 g ha −1 glufosinate plus 20 g L −1 ammonium sulfate (AMS). Untreated plants were sprayed with AMS only, and samples were collected at 24 h after treatment (HAT). Leaf tissue (200 mg) was ground in liquid nitrogen with a mortar and pestle. The powder was homogenized with 10 ml methanol-water (75:25 [v/v]) and incubated in ultrasonic bath for 30 min before being centrifuged at 4,000 × g for 10 min. The supernatant (1.5 ml) was filtered through a 0.2 μm nylon filter into an UHPLC vial, and 1 μl was injected for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis (Shimadzu Scientific, Columbia, MD, USA).

Isobole Analysis
Dose-response experiments were conducted for both the herbicides glufosinate and saflufenacil to estimate the dose for 50% reduction in dry shoot biomass (ED 50 ). The doses were 0,9,17,35,70,140,280, and 560 g ha −1 for glufosinate and 0, 0.5, 1, 2, 4, 8, 16, and 32 g ha −1 for saflufenacil. Each treatment had three replications, and the same experiment was conducted twice. The variable response was shoot dry biomass (percentage relative to untreated plants at 21 d after treatment [DAT]). An isobole shows dose combinations of two herbicides providing the same effect. For example, if x g ha −1 of glufosinate or y g ha −1 of saflufenacil individually provided 50% injury, then 1/2x glufosinate þ 1/2y saflufenacil should also provide 50% injury. However, if the effect was synergistic or antagonistic, the output should be different than 50% injury. We tested five dose responses with different mixture ratios ( Table 1) that were chosen based on the ED 50 values obtained with the two herbicides tested individually. Then, the proportion of each herbicide required to achieve the ED 50 was plotted in a scatter graph. An extension of the additive model was used to explain the interaction between the two herbicides and predict the shape of the isobole (Sørensen et al. 2007). If the shape is linear, the mixture effect is additive. If the curve is convex, the two herbicides are antagonistic to each other. Finally, a concave shape indicates that the mixture is synergistic (Streibig et al. 1998).

Response of Protoporphyrinogen Oxidase-Resistant Amaranthus tuberculatus and Glufosinate-Resistant Soybean to Glufosinate and PPO Inhibitors
To investigate whether glufosinate enhances PPO inhibitors or PPO inhibitors enhance glufosinate, we tested a PPO-resistant A. tuberculatus biotype and a glufosinate-resistant soybean variety ('S120090', Liberty Link TM , Pioneer, IA, USA). The resistance mechanism in the PPO-resistant A. tuberculatus biotype had been previously described as a codon deletion in PPO2 (ΔG210), providing 23-fold resistance to lactofen, and the seeds were collected in Adams County, IL (Dayan et al. 2010;Patzoldt et al. 2006). A glufosinate-susceptible soybean variety ('P0007A43R', Pioneer, IA, USA) and a PPO-susceptible A. tuberculatus biotype from Nebraska were also included for comparison. Plants were grown as described earlier and 6-leaf-stage plants were sprayed with half the rate of glufosinate (280 g ha −1 ), low dose (<5% field rate) of PPO inhibitor (lactofen at 5 g ha −1 for A. tuberculatus, and saflufenacil at 1 g ha −1 for soybean), or the tank mixture of the two herbicides for each plant species. Lactofen was tested on A. tuberculatus, because the resistance level was higher for this herbicide compared with saflufenacil. Three replicates were evaluated, and the experiments were conducted twice. Response variables were visual injury (scale 0 to 100, where 0 means no effect and 100 implies plant death), ROS accumulation, and proto levels for A. tuberculatus; and visual injury, proto levels, and glufosinate and glutamate concentration for soybean. Glufosinate and glutamate were quantified over time up to 24 HAT, whereas other variables were evaluated at 12 HAT only. Hydrogen peroxide (H 2 O 2 ) and superoxide (O 2 − ) were measured by staining leaf disks in solutions containing DAB and NBT, respectively. The DAB solution contained 0.1 g DAB solubilized in 200 ml water at pH 3.8. The NBT solution was composed of 0.1 g NBT, 13.6 g potassium phosphate monobasic, and 1.3 g sodium azide in 200 ml water. Sixteen leaf disks (5-mm diameter) from each treatment were placed in 20-ml glass tubes containing each staining solution. The samples were then shaken under 67 kPa vacuum for 1 h. Leaf disks were washed in distilled water and boiled in 70% (v/v) ethanol, which was replaced every 20 min for four cycles. Leaf disks were stored in 70% (v/v) ethanol for 12 h and scanned (Brother DCP-L2550DW, Bridgewater, NJ, USA). The levels of H 2 O 2 or O 2 − were quantified using CS3 Photoshop (Adobe Systems, San Jose, CA, USA) by measuring the color intensity in each leaf disk and removing background levels (Dayan et al. 2019a;Mendes et al. 2020;Takano et al. 2019). Data were represented as relative intensity of treated samples compared with untreated samples (treated intensity − untreated intensity).
Glufosinate and glutamate were extracted and quantified as previously described. Proto extraction and analysis followed a protocol described elsewhere (Dayan et al. 2015). Leaf tissue (0.2 g) was ground to a powder in liquid nitrogen and homogenized in 2 ml methanol:0.1M NH 4 OH (9:1) and centrifuged at 10,000 × g for 15 min. The supernatant was saved and the pellet rehomogenized in 1 ml methanol:0.1M NH 4 OH (9:1), then centrifuged again at 10,000 × g for 15 min. Supernatants were pooled and then filtered through a 0.25-μm nylon syringe membrane filter before quantification with the LC-MS/MS system described earlier. Metabolites were separated in a biphenyl column (100 by 4.6 mm, 2.6 μm, 40 C) at a flow rate of 0.4 ml min −1 using a linear gradient of methanol (B) and 10 mM ammonium acetate (A): 0 min, 50% B; 8 min, 70% B; 11 min, 90% B; 13 min, 90% B; 13.5 min, 50% B; 17 min, 50% B. The MRM was optimized to 340.10 > 227.95 (Moulin et al. 2008). Standard curves generated with serial dilutions of authentic standards were used for quantification. respectively. Weed density was assessed by randomly counting the number of plants in 1 m 2 within each plot before herbicide application. The soil type was Satanta loam (fine-loamy, mixed, superactive, mesic Aridic Argiustolls) with 3.5% organic matter and 7.5 pH in the top 20 cm. Plots were 3-m wide and 7-m long with the central 18 m 2 used for evaluations. Glufosinate (420 g ha −1 ) was applied alone or in tank mix with different PPO inhibitors (g ha −1 ): flumioxazin (2.5), saflufenacil (1), pyraflufen-ethyl (0.2), lactofen (4.2), and fomesafen (7.1). Methylated soybean oil (0.5% [v/v]) was added to all treatments, which were sprayed using a CO 2 -pressured backpack sprayer attached to a boom equipped with six flat spray nozzles (TeeJet®, Louisville, KY, USA) spaced 0.5 m from each other. Pressure was maintained at 25 kPa and speed at 3.2 km h −1 , providing 160 L ha −1 spray volume. Visual injury (%) was visually graded at 21 DAT using a scale where 0 means no symptoms and 100 means death of all plants (Frans 1986).

Efficacy of Glufosinate + Saflufenacil under Low Temperature and Humidity
Plants of A. palmeri at the 6-leaf stage were incubated under either 25 C and 70% RH or 13 C and 30% RH for 24 h. Plants were sprayed with glufosinate (280 g ha −1 ), saflufenacil (1 g ha −1 ), or glufosinate þ saflufenacil (280 þ 1 g ha −1 ) and immediately returned to their respective environmental conditions. Visual injury was evaluated at 14 DAT using the same method previously described.

Data Analysis
All experiments were repeated in time. Graphs were created using Prism software (GraphPad v. 8.3.1, San Diego, CA, USA). Statistical analysis was performed with R software (R Studio v. 3.6.1, Vienna, Austria). A four-parameter log-logistic regression was fit for the dose-response experiments considering the goodness-of-fit values and the fact that it is by far the most used model for this type of data set (Ritz and Streibig 2014). Data normality and homogeneity of variance were checked by Q-Q plots and residuals versus fitted values, respectively. Treatments were considered fixed effects, and greenhouse experiments were conducted in a complete randomized design, while field experiments were conducted in a randomized complete block design. Differences

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Takano et al.: Glufosinate þ PPO inhibitors between the two treatments were compared by t-test (P < 0.05).
Data from experiments with more than two treatments were subjected to ANOVA, and means were compared using Tukey's test (P < 0.05). The isobole analysis was performed based on previous literature (Sørensen et al. 2007;Streibig et al. 1998).

Results and Discussion
Amino Acid Levels Inhibition of GS by glufosinate depleted the levels of glutamine and glutamate, but increased proline and arginine at 24 HAT ( Figure 1A). In most biochemical reactions, when the enzyme is inhibited, the substrate tends to accumulate, whereas the product is normally depleted. For instance, glyphosate inhibition of 5-enolpyruvylshikimate-3-phosphate synthase leads to the accumulation on shikimate to high levels (Shaner et al. 2005). Thus, glutamine levels are expected to decrease and glutamate to accumulate following glufosinate treatment. Instead, both glutamate and glutamine were depleted at 24 HAT. However, when glutamate levels were tracked over time after glufosinate treatment, a transient accumulation was observed ( Figure 1B). Up to 3 HAT, glutamate levels were significantly higher in treated plants compared with the untreated plants. Therefore, glufosinate induces a transient accumulation of glutamate in A. palmeri.

Isobole Analysis
The ED 50 values for glufosinate and saflufenacil in A. palmeri were 50 and 2.5 g ha −1 , respectively (Figure 2A and B). These doses were used in the isobole analysis to evaluate the interaction between these two herbicides ( Figure 2C; Supplementary Figure S1). Lower ED 50 values were obtained with the mixture compared with the herbicides used individually. This means that lower mixture doses were required to provide the same effect of the two herbicides individually. The concave shape of the isobole indicates strong synergism between glufosinate and saflufenacil. In addition to A. palmeri, we also tested the effect of glufosinate and PPO inhibitors ( Figures S2 and S3). In general, the enhanced activity with the mixture was more evident for broadleaf than grass species.

Effect of Glufosinate + Lactofen on Amaranthus tuberculatus resistant to PPO Inhibitors
Half-rate of glufosinate (280 g ha −1 ) provided 20% injury on both PPO-susceptible and PPO-resistant A. tuberculatus at 12 HAT ( Figure 3A-C). An ultra-low dose of lactofen (5 g ha −1 ) caused less than 5% injury on both biotypes. Conversely, the herbicide mixture provided 94% injury to the susceptible biotype, but only 20% injury to the resistant one. These results were consistent with the accumulation of ROS ( Figure 3D). H 2 O 2 and O 2 − accumulated to high levels in the susceptible biotype treated with glufosinate þ lactofen. Low levels of these free radicals were observed in both biotypes after glufosinate treatment, similar to the effect of the herbicide mixture on the PPO-resistant biotype. In contrast, proto levels increased up to 19 nmol g −1 in the susceptible biotype treated with glufosinate þ lactofen. Small quantities (<1 nmol g −1 ) were detected in the PPO-susceptible biotype treated with lactofen alone and in the PPO-resistant biotype treated with the herbicide mixture. Proto did not accumulate in either biotype treated with glufosinate. These results suggest that glufosinate enhances lactofen activity by increasing the accumulation of proto. Complementary data obtained with A. palmeri corroborated these findings (Supplementary Figure S2).

Effect of Glufosinate + Saflufenacil on Glufosinate-Resistant Soybean
Glufosinate (280 g ha −1 ) significantly caused more injury to glufosinate-susceptible (37%) than glufosinate-resistant (5%) soybean ( Figure 4A). A very low dose of saflufenacil (1 g ha −1 ) provided less than 3% injury to both soybean varieties. The combination between these two herbicides, however, caused high levels of injury to glufosinate-resistant (68%) and even more to glufosinatesusceptible (82%) soybean. These injury levels provided by saflufenacil alone or in combination with glufosinate are consistent with proto accumulation ( Figure 4B). Proto levels were very low (<0.5 nmol g −1 ) in plants treated with saflufenacil only. The addition of glufosinate to saflufenacil enhanced proto accumulation in both varieties, especially for the glufosinate-susceptible soybean.
Glufosinate levels increased over time in the glufosinatesusceptible soybean following treatment ( Figure 4C). In contrast, glufosinate-resistant soybean had increased glufosinate levels until 4 HAT and levels decreased after that, due to the metabolism into N-acetyl-L-phosphinothricin by the acetyltransferase activity. This spike in glufosinate levels at 4 HAT was likely enough to cause some GS inhibition and a transient accumulation of glutamate even for glufosinate-resistant soybean ( Figure 4D). Glutamate levels significantly increased (2-to 3-fold) after glufosinate treatment in soybean. These results support the hypothesis that glufosinate synergizes PPO inhibitors by increasing the levels of glutamate, which is diverted into the accumulation of proto in the chlorophyll biosynthesis pathway.

Field Performance of Glufosinate + PPO Inhibitors on Bassia scoparia
Glufosinate (420 g ha −1 ) provided 25% injury on B. scoparia glufosinate alone, with no statistical differences (P < 0.05) among them. The addition of any PPO inhibitor to glufosinate significantly increased injury levels compared with glufosinate only. Glufosinate þ saflufenacil provided the highest levels of injury on B. scoparia but did not differ from glufosinate þ pyraflufen. These results support our findings obtained in the greenhouse and in the lab, demonstrating the enhanced activity with glufosinate þ PPO inhibitors compared with the two herbicides applied individually.

Saflufenacil Alleviates the Lack of Efficacy by Glufosinate under Low Temperature and Low Humidity
Both glufosinate (280 g ha −1 ) and glufosinate þ saflufenacil (280 þ 1 g ha −1 ) provided 100% injury to A. palmeri under 25 C and 70% RH ( Figure 6A) at 14 DAT. Glufosinate efficacy decreased to 60% under 13 C and 30% humidity. Less than 10% injury was obtained with saflufenacil under both conditions. The combination between glufosinate and saflufenacil, however, still provided 100% injury, even under low temperature and low relative humidity. These results suggest that a low dose of saflufenacil can help to overcome the negative effect of low temperature and humidity on glufosinate efficacy.
In the light of an increased number of multiple herbicideresistant weeds, enhancing the efficacy of alternative tools like glufosinate is needed. Glufosinate performance can vary in the field, because it does not translocate well in plants (Beriault et al. 1999;Everman et al. 2009;Kumaratilake et al. 2002;Takano et al. 2020b). Thus, we took a biochemical approach to enhance the activity of glufosinate. Both glufosinate and PPO inhibitors are fast-acting herbicides due to the accumulation of ROS and therefore could enhance each other's activity. The isobole analysis confirmed our hypothesis that the mixture glufosinate þ saflufenacil is synergistic and stronger for glufosinate enhancing PPO inhibitor than PPO inhibitor enhancing glufosinate ( Figure 2C). PPO inhibitors target chlorophyll biosynthesis by  . The graph shows the proportion of each herbicide to achieve 50% reduction in dry biomass (ED 50 ). If no synergism had been observed for the two compounds, data points were expected to fall into the red dashed line (additive effect). The concave shape of the isobole curve indicates high levels of synergism between the two herbicides. Error bars represent the standard deviations of the means.
inhibiting PPO, a key enzyme in the pathway (Figure 7). Inhibition of PPO leads to proto accumulation, the product of the reaction catalyzed by the enzyme (Duke et al. 1991). The accumulation of proto is toxic, because it can absorb light energy and transfer to molecular oxygen, generating ROS and causing lipid peroxidation followed by cell death (Dayan et al. 2019b). The biochemical basis for the synergism observed with glufosinate þ saflufenacil relies on a change in glutamate flux by glufosinate, driving this amino acid toward chlorophyll synthesis, resulting in enhanced accumulation of proto with the addition of the PPO inhibitor (Figure 7). The synergism effect is stronger for glufosinate enhancing the activity of PPO inhibitors than the reverse. This is supported by the fact that PPO-resistant A. tuberculatus plants were not killed with the herbicide mixture and showed similar injury levels when glufosinate was sprayed alone (Figure 3). Glutamate, the substrate for GS, transiently accumulates shortly after glufosinate treatment, and it is diverted into other metabolic pathways including proline, arginine, and chlorophyll biosynthesis (Figures 1, 4, and 7). Proline and arginine accumulate after GS inhibition, supporting our hypothesis.
Glufosinate-resistant soybean express the pat gene and metabolize glufosinate by adding an acetyl group to the herbicide molecule (Dröge et al. 1992;Strauch et al. 1988). Even though these plants are resistant to the herbicide, glufosinate concentration in the leaf tissue increased over the first 4 HAT and caused partial GS inhibition ( Figure 4A). This is because uptake is probably faster than metabolic rates during the first few hours after treatment, leading to glufosinate accumulation (Carbonari et al. 2016). Consequently, glutamate levels also increased up to 4 HAT, enhancing proto accumulation in the presence of saflufenacil (Figure 4). An unknown compound with the same mass as glutamate-1-semialdehyde and glutamate-4-semialdehyde accumulates at high levels in glufosinate-treated plants (Supplementary Figure S4). These two metabolites are precursors for chlorophyll and proline/arginine biosynthesis, respectively, supporting the fact that glufosinate synergizes PPO-inhibitor activity by a transient glutamate accumulation. Proto accumulation at high levels leads to ROS generation and the catastrophic consequences of lipid peroxidation (Dayan et al. 2019b). To evaluate the applicability of the herbicide mixture, we tested glufosinate in combination with low doses of different PPO inhibitors to control B. scoparia in the field. A tank mix with PPO inhibitors provided increased injury levels compared with glufosinate only ( Figure 5). The highest levels of injury were obtained with glufosinate þ saflufenacil, similar to levels seen with glufosinate þ pyraflufen-ethyl. While glufosinate efficacy can be strongly affected by low temperature and low humidity (Coetzer et al. 2001;  Figure 6. Saflufenacil (Saflu) at 1 g ha −1 can overcome the lack of efficacy by glufosinate (Gluf) at 280 g ha −1 on Amaranthus palmeri under low temperature and humidity. Visual injury provided with glufosinate þ saflufenacil compared with the two herbicides applied individually at high (A) and low (B) temperature and humidity. Means with the same letter do not differ according to Tukey's test (P < 0.05). Error bars represent the standard deviations of the means. Glutamate is the precursor for the biosynthesis of glutamine, arginine, proline, and chlorophyll. Inhibition of glutamine synthetase (GS) diverges the fate of glutamate into proline/arginine and chlorophyll biosynthetic pathways. Inhibition of both GS and PPO leads to increased protoporphyrin accumulation and subsequent formation of reactive oxygen species (ROS). 330 Takano et al.: Glufosinate þ PPO inhibitors Kumaratilake and Preston 2005), this can be overcome with the addition of a low dose of saflufenacil to the tank ( Figure 6). Future research will investigate potential opportunities to mitigate resistance to PPO inhibitors with the herbicide mixture. Furthermore, while the tank mix of glufosinate and PPO inhibitors seems to be very promising for burndown applications, it was not safe for glufosinateresistant soybean POST. However, if used on crops tolerant to both glufosinate and PPO inhibitors, this approach could offer new opportunities for better weed control, especially where resistant weeds are present. In this study, we used reduced doses of both glufosinate and PPO inhibitors because we wanted to prove the concept. Under real-world circumstances, full doses of the herbicides must be tested, especially when managing resistant weeds. Therefore, more data are needed to better understand the most appropriate uses for this herbicidal composition as well as potential technical adjustments (e.g., doses, active ingredients, weed species, cropping systems). The combination of glufosinate and PPO inhibitors provided enhanced herbicidal activity compared with the products applied individually. Inhibition of GS by glufosinate leads to a transient accumulation of glutamate, the precursor for chlorophyll biosynthesis in plants. Inhibition of chlorophyll biosynthesis by PPO inhibitors causes accumulation of proto, which is enhanced in the presence of glufosinate. Thus, the synergism observed with the mixture results from proto accumulation to higher levels when both GS and PPO are inhibited simultaneously. The herbicide combination provided improved weed control in the field and may help to overcome negative effects of environmental conditions on glufosinate performance.


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Performance of Blast and Drought Tolerant Finger Millet Variety ML-365 under Front Line Demonstrations in Tumkur, Karnataka

Finger millet (Eleusine coracana L. Gaertn) is one of the important millets grown extensively in Tumkur District. It is a hardy crop, has good adaption to wide range of environment especially heat, drought, marginal and degraded soils (Okalebo et al., 1991). It is mainly grown for its grains and it is highly nutritious. Its grains contain carbohydrate (65-75%), protein (5-8%), dietary fibre (15-20%), minerals (2.5-3.5%) and vitamins (Chethan and Malleshi, 2007). It is superior to rice and wheat, in respect of crude fibre, amino acids and minerals like calcium (344 mg/100g) and potassium (408 mg/100g). It also contains anti nutrients such as phytates, polyphenols, tannins and trypsin inhibitory factors.

Introduction
Finger millet (Eleusine coracana L. Gaertn) is one of the important millets grown extensively in Tumkur District. It is a hardy crop, has good adaption to wide range of environment especially heat, drought, marginal and degraded soils (Okalebo et al., 1991). It is mainly grown for its grains and it is highly nutritious. Its grains contain carbohydrate (65-75%), protein (5-8%), dietary fibre (15-20%), minerals (2.5-3.5%) and vitamins (Chethan and Malleshi, 2007). It is superior to rice and wheat, in respect of crude fibre, amino acids and minerals like calcium (344 mg/100g) and potassium (408 mg/100g). It also contains anti nutrients such as phytates, polyphenols, tannins and trypsin inhibitory factors.
Regular consumption of whole grain of finger millet and its products helps in managing International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 9 Number 12 (2020) Journal homepage: http://www.ijcmas.com Frontline demonstrations were conducted in farmers' fields of Tumkur district of Karnataka, India during kharif 2016 to 2018 to create awareness among the farmers and demonstrate the improved production technologies in finger millet. The integrated crop management practices including cultivation of drought and blast tolerant finger millet variety ML 365, integrated nutrient management, integrated pest and disease management practices were demonstrated and compared with the existing farmers practice followed in Finger millet cultivation. Results showed that demonstration of finger millet variety ML 365 with integrated crop management practices recorded higher grain yield of 23.61q/ha and farmers practice recorded lower yield of 18.46 q/ha. Adoption of integrated crop management practices increased the grain yield of finger millet to the tune of 28.16 per cent compared to farmers practice. Farmers earned higher net income of Rs.34661/ha through the demonstration and Rs.21428/ha with farmers practice. Besides, farmers realized higher benefit cost ratio (2.66) through the demonstration compared to farmer's practice (2.10). Thus, the frontline demonstration of improved variety with crop management practices increased the grain yield and net income of the farmers growing finger millet under rainfed condition. In the present study, potential of the improved variety and technologies were demonstrated systematically and scientifically in the farmers field along with farmers practice for further adoption by farming community in large scale. Article Info diabetes and its complications by regulation of glucose homeostasis and prevention of dyslipideamia. It also gives protection against the risk of cardiovascular disease, gastrointestinal cancers and other health issues. It has health beneficial effects, such as anti-diabetic, anti-diarrheal, antiulcer, antiinflammatory, antitumerogenic, atherosclerogenic effects, antioxidant and antimicrobial properties (Devi et al., 2014). Hence, there is a great demand for improving finger millet production.

K e y w o r d s
Finger millet is being cultivated in an area of about 1,85,000 hectares in Tumkur District. About 90 per cent of the area under Finger millet is being cultivated under rainfed condition during kharif season. Under rainfed condition, farmers facing the problem of moisture stress at various crop growth stages thereby experiencing low yield and crop loss to some extent. Besides moisture stress, lack of knowledge on the availability of drought tolerant varieties, non adoption of improved cultivation practices, prevalence of nutrient deficiency, pest and disease incidence also lowers the finger millet productivity.
Hence, the productivity of finger millet might be increased by growing suitable variety along with improved crop management practices. Similar studies on crop yield increase by adoption of improved crop management practices were reported by Subhashree et al., (2017) in Finger millet; Sharma et al., (2016) and Singh (2017) in Wheat; Jat and Gupta (2015) in Pearl millet; Meena et al., (2014) in Maize Considering the above facts, a frontline demonstration was proposed and conducted in the farmers' holdings to demonstrate the improved package of practices for higher productivity in finger millet under rainfed condition.

Materials and Methods
Frontline demonstration was conducted to demonstrate the potential of the drought and blast tolerant variety with the improved package of practices in comparison with the existing farmers practice in the farmers' holdings of Tumkur district during kharif 2016-2018 under rainfed condition. Demonstration was conducted in 25ha area in three villages involving 50 farmers. The soils of the demonstration fields were collected and analysed for its initial soil nutrients status. The results showed that the soils were slightly alkaline in soil reaction, non saline, low in nitrogen, medium in phosphorus and potassium nutrient content. Each demonstration was conducted in an area of 0.4 ha and with an adjacent area of 0.4 ha selected for farmers practice. In the demonstration, the improved practices including cultivation of finger millet variety ML 365, integrated nutrient management, integrated pest and disease management practices were demonstrated along with the farmers practice.
Finger millet variety ML 365 was released from University of Agricultural Sciences, Bengaluru during 2008. It has 100-105 days duration, high yielding variety, tolerant to drought and blast disease. In farmers practice, finger millet variety GPU 28 was grown with the existing farm-ers practices such as broadcasting of seeds, basal application of complex fertilizers, etc. The details on the technological interventions followed in the demonstration and farmers practice were given in Table 1. Before initiating the demonstration, the beneficiary farmers were trained in all the improved practices in finger millet cultivation and followed in the demonstrations. Demonstration field were periodically observed by the scientists of Krishi vigyan Kendra.
At the time of harvest, the data on plant population (number), plant height (cm), number of tillers per plant (number), days taken for 50% flowering (number) and grain yield (q/ha) of finger millet crop were recorded from both the demonstration and farmers practice. Based on the cost of inputs and market price of the produce, economic parameters such as net return (Rs/ha) and benefit cost ratio were worked out.

Results and Discussion
Results of the study indicated that demonstration of drought and blast tolerant finger millet variety ML 365 with integrated crop management practices recorded the higher plant population (35.5/m2), plant height (75.0 cm) and higher number of tillers per plant (4.55). Lower plant population (28.5/m2), plant height (64.5 cm) and number of tillers per plant (2.5) were recorded in farmers practice ( Table 2). The demonstrated variety attained maturity one week earlier than the existing local variety. Cultivation of drought tolerant finger millet variety ML 365 with integrated crop management practices recorded higher average grain yield of 23.61q/ha (Table 3). Farmers practice recorded lower average grain yield of 18.46q/ha. Adoption of improved practices increased the yield of finger millet to the tune of 28.16 per cent compared to the farmers practice under rainfed condition. The increased yield under demonstration might be due to the combined effect of high yielding, drought tolerant variety and adoption of improved crop management practices. The similar results of yield enhancement through front line demonstration of improved technologies has been reported by Kumar et al., (2010) in bajra; Solanki et al., (2014) in maize and Anand Naik et al., (2016) in sorghum. Besides, the incidence of blast disease was not reported in the demonstrated variety and it was 8 per cent in the farmers practice.  . It might be due to the higher grain yield recorded in demonstration compared to farmers practice. Similar results of increase in net income and benefit cost ratio due to adoption of improved technologies in the demonstrations were reported by Jat and Gupta (2015) in pearl millet; Dhaka et al., (2010) in maize and Anand Naik et al., (2016) in sorghum.
Results of the demonstration revealed that cultivation of finger millet variety ML 365 with integrated crop management practices increased the yield and income of the farmers under rainfed condition. In addition, the introduced variety has satisfied the farmers preferences such as high tiller production, early maturity and tolerance to grain shattering or dusting.
Hence, the farmers were convinced with the performance of the variety with regard to its yield potential and tolerance to biotic and abiotic stresses under rainfed condition.


text:
Effect of Bentonite on the Early Growth Characteristics of Teak Seedlings Planted in Sandy Soil in Northeast Thailand—A Pilot Study

: The growth of teak ( Tectona grandis L. f.) seedlings in sandy soil in northeast Thailand is suppressed by infertility and drought stress. In a preliminary ﬁeld pot experiment, we conﬁrmed that bentonite was useful for increasing soil water availability. To monitor early growth characteristics of teak seedlings in sandy soil, we curried out an experiment of teak seedlings using bentonite and fertilizer in the ﬁeld from July 2014 to November 2015. We then compared the growth, biomass, photosynthetic rate, leaf water potential, and concentration of elements in the plant organs among the four treatments. Bentonite increased both soil pH and Ca during the experimental period, and retained P in fertilizer. The dry mass of teak seedlings was markedly increased by concurrent use of bentonite and fertilizer. However, the use of bentonite alone showed little increase of dry mass of teak seedlings. Moreover, application of bentonite was mitigated drought stress in dry season, and photosynthetic rate showed high value by high concentration of chlorophyll. The concentration of K in root of teak seedlings was increased by application of bentonite, and its value showed positive correlation with the relative growth rate of teak seedlings. We concluded that bentonite was useful for the uptake of K and retention of P in fertilizer, as a result, growth of teak seedlings was accelerated.

Introduction
Teak (Tectona grandis L. f) is one of the most important timber species, and has been planted in tropical regions of various countries [1]. In general, natural teak forest soil is alkaline (pH 6.8-7.8) [2], and its area in Thailand overlaps with the distribution of calcareous soil originating from limestone [3]. Soil nutrients are important in the growth of teak seedlings, and limited soil nutrient content is a primary restriction on the growth of teak [1,[3][4][5]. In particular, a drastic decrease in the growth of teak seedlings has been observed when they are raised in culture medium without nitrogen [N] or calcium [Ca] [4,6]. Thus, we considered N and Ca to be essential nutrients for teak growth.
In the uplands of tropical Asia, soil is characterized by low pH and infertility [7][8][9]. Moreover, clays at the surface have been leached, and the water-holding capacity and cation exchange capacity (CEC) at the surface are low [8,9]. The growth of teak is negatively correlated with sand content, and poor teak growth is observed on plantations with very sandy soil [3,10,11]. Additionally, soil with a low CEC is characterized by rapid leaching after fertilizer application [9,12]. Soil in northeast Thailand is described as a "light textured sandy soil" (referred to as sandy soil herein), and is characterized by low clay content, low pH, low fertility, low water-holding capacity, and low CEC [13][14][15][16]. Previous studies have shown that teak grows poorly in sandy soils [17] and readily suffers from drought stress [15]. Thus, when teak seedlings are planted in sandy soil, the water-holding capacity and CEC must be improved to promote growth.
Meanwhile, there was a case that height of teak planted in northeast Thailand was over 10 m for five years by soil improvement [16]. This value was similar with the suitable region of teak plantation in Thailand [18]. Thus, some areas in northeast Thailand have a potential for high growth of teak [19]. Moreover, growth characteristics of teak seedlings for two years was reflected subsequent growth [11,16]. Therefore, the monitoring of teak growth in early period after planting is important [15,20], and can predict the subsequent growth [11,16]. Moreover, teak seedlings are suffered from the drought stress in the first dry season, and increase their mortality [15,20]. Measurements of leaf water potential and photosynthetic rate can estimate the vitality of teak seedlings [21][22][23]. Therefore, measurements of ecophysiological traits of teak seedlings in early period is essential for the monitoring of teak growth.
The previous method for the improvement of sandy soil was fertilization and acid correction [16]. Recently, bentonite, a clay consisting mostly of montmorillonite is introduced, and has a high water-holding capacity and CEC [13,[24][25][26]. However, the use of bentonite in silviculture is rare [24,27]. In a previous experiment, we examined the effects of bentonite on teak growth [14]. We added 4% bentonite to sandy soil in northeast Thailand. Teak seedlings were potted in these soils and raised for 1 year under field conditions with no fertilization. Soil water content was increased by adding bentonite; however, we did not confirm growth acceleration in the teak seedlings after the addition of bentonite [14]. In contrast, application of bentonite improves the fertility of sandy soil owing to its high CEC value. A particle of bentonite is charged negatively, and bind various kind of cations [28]. As a result, bentonite has high capacity of nutrient retention, and plants efficiently absorb cations bound a surface of bentonite [29]. Moreover, application of bentonite has the capacity to increase Ca in the soil [26]. Bentonite has also a capacity to mitigate the leaching of nutrient ions by binding on its surface [29]. However, the capacity of binding is not irreversible [30]. Plant production is increased by concurrent use of bentonite and fertilizer [29,31]. However, verification tests on teak seedlings grown in bentonite and fertilizer-enhanced soils have not yet been conducted in a field environment.
The aim of this study was to examine the suitability of bentonite for improving the CEC and fertility of sandy soil. We conducted a field experiment with teak seedlings grown in sandy soil containing added bentonite and fertilizer. We hypothesized that the concurrent use of bentonite and fertilizer would increase the soil's capacity for retention of nutrients in fertilizer and that the growth of teak seedlings should be accelerated by efficient nutrient uptake, and mitigate drought stress in dry season. To verify these hypotheses, we examined the ecophysiological traits of teak seedlings in early growth period, including (1) soil nutrient content, (2) seedling growth and biomass, (3) characteristics of photosynthesis and water use, and (4) concentrations of elements in plant organs. These parameters were compared among seedlings grown in untreated sandy soil, grown with a single use of fertilizer or bentonite, and grown with concurrent use of fertilizer and bentonite. In addition, symbiosis of arbuscular mycorrhizal (AM) fungi is important to the growth of teak seedlings [32]. We also observed colonization of AM fungi in the roots of the teak seedlings.

Study Site
We curried out the experiment at the Northeast Forest Seed Center, which is located in Khon Kaen Province in northeastern Thailand (16 • 16 N, 102 • 47 E, 191 m a.s.l.). The metrological data of this center was introduced by Kayama et al. [14]. Precipitation is concentrated from May to October, and other months are considered as dry season [14]. The sandy soil in this area is classified as an Acrisol in the FAO/Unesco soil taxonomy, and as an Ultisol in the USDA taxonomy [7,8].
On the selection on the suitable site for the planting of teak seedlings, we already estimated from the soil research, and categorized as five ranks [19]. Sandy soil is categorized third rank, and is considered as moderately suited soil [19]. Sandy soil is distributed over a large area in this province (13.5%) [19], and there is no natural teak forest in the sandy soil area. Farmers living in this area are interested in the cultivation of teak for its high timber quality [33]. Soil improvement is essential to accelerate the growth of teak planted in sandy soil area.

Selection of Experimental Plots and Preparation of the Land and Teak Seedlings
A sandy soil experimental plot, which covered 2016 m 2 , was selected as the site for our experiment. This site had been previously planted with teak seedlings under nofertilization conditions [17]; however, many of these teak trees were dwarfed. We prepared the land in April 2014. All trees growing at this site were cut down, and their roots were removed.
We prepared teak seedlings by a tissue culture technique at the Ngao Silvicultural Research Station, Lampang, Thailand, and 520 teak seedlings were transported to the Northeast Forest Seed Center. The number of seedlings included was sufficient to replant the study area. The teak clone was selected from Mae Hong Son Province (clone number 21), and this clone has been planted in various locations of Thailand (Royal Forest Department).

Experiment
The following treatments were established: control (CON), fertilizer (FER), bentonite (BEN), and bentonite and fertilizer (BEN + FER). Three blocks of four treatments were established at the site, and their positions were randomized. The size of each block was 12 m × 10 m, with 2 m between blocks. Teak seedlings were planted as a buffer on the border between the blocks. After establishing the seedlings, we buried a concrete pole at the corner of each block.
We prepared the bentonite and fertilizer before planting. In June 2014, we prepared 120 kg sodium [Na] bentonite and 12 kg of chemical fertilizer (N: phosphorus [P]: potassium [K] = 15:15:15), which was purchased from a market in Thailand. After preparing these materials, 1 kg of bentonite was placed in separate plastic bags, and 100 g fertilizer was added to each plastic bag.
We planted 42 teak seedlings in each replicate block at the site during July 2014. The total number of teak seedlings planted was 504 (4 treatments × 3 replicates × 42 seedlings/ replicate). The interval between seedlings was 2 m × 2 m, and 22 teak seedlings were planted outside the blocks as buffer trees. We targeted 20 teak seedlings per replicate block planted within the buffer trees. Before planting, we made a hole at the position of the teak seedling. The area of hole was 30 cm × 30 cm, and 30 cm of depth. The removed soil was collected, and 1 kg of bentonite or 100 g of fertilizer was mixed into it. The amount of fertilizer was the same as that used in a previous fertilization test in sandy soil [16]. After mixing, a teak seedling was planted in this hole and buried in the mixed soil. We also sampled 10 teak seedlings at this time. We collected 2 g of fine roots with diameters <2.0 mm and placed them in a solution of formaldehyde, acetic acid, and alcohol (FAA solution), where they were preserved at 4 • C. These samples were used to observe AM colonization by the following method. Other teak seedling organs were oven-dried at 70 • C for 3 days, and the dry masses of the leaves, stems, and roots were recorded.
We also inserted soil moisture sensors (SM150, Delta-T Devices Ltd., Cambridge, UK) into two blocks. The sensor logger was fixed to a concrete pole, and a soil sensor was inserted near the seedling.

Soil Analysis
We measured the texture, the CEC and chemical properties of sandy soil. On the subject of chemical properties, we analyzed pH, organic matter (OM), total N, exchangeable p and base cations (Ca, Magnesium [Mg], K and Na). We selected three seedlings from each replicate block located at three corners and collected surface (0-5 cm) soil samples under these seedlings in July 2014 and November 2015. For the measurement of soil pH, we prepared the suspended solution by addition of 10 g of fresh soil and 25 mL of distilled water, and mixing for 1 h [34]. After mixing, Soil pH was measured using a pH meter (SG2, Mettler Toledo, Zürich, Switzerland). The soil samples were air-dried prior to the chemical analysis.
The analysis of soil texture was used for the samples in July 2014, and determined by the hydrometer method [35]. The extraction solution for the CEC analysis was obtained by the rapid method [36] produced by Fujihira Industry Co. (Field Soil Doctor, Tokyo, Japan). The ammonia concentration of this solution was analyzed by the indophenol method [37] using a spectrophotometer (AE-VIS721, A and E Lab. Co., London, UK), and then the CEC value was calculated. The OM concentration was determined by the loss-onignition method [38]. Total N concentration was determined by the Kjeldahl method [38]. Exchangeable p was obtained using dilute acid fluoride [38] and shaking for 1 min. p in the extracted solution was analyzed by the molybdenum blue method [39] using a spectrophotometer (U-1800, Shimadzu, Kyoto, Japan). Exchangeable base cations were obtained by mixing 4 g of dry soil with 100 mL of 1 M ammonium acetate solution and shaking for 1 h [38]. The base cations in the extracted solutions were determined using an atomic absorption spectrophotometer (AAnalyst 300, Perkin-Elmer, Norwalk, CT, USA).

Measurement of Growth of Teak Seedlings
We measured the growth of 20 teak seedlings for three blocks of four treatments. The total number of seedlings was 60 for each treatment. Tree height and basal diameter were measured at six time points (July 2014, October 2014, February 2015, May 2015, July 2015, and November 2015). At these times, we also confirmed the number of dead teak seedlings.

Measurement of Photosynthetic Rate and Concentrations of Chlorophyll
We measured the area-based photosynthetic rate at light saturation (P sat ) and stomatal conductance (gs) in healthy teak leaves at the second level from the top. Some teak seedlings had only immature or senescent leaves by short period of leaf turnover [14]. To unify mature and healthy teak leaves, we selected 9 teak seedlings from 20 seedlings (three individuals from each of three blocks), were used to measure the photosynthetic rate. We measured the photosynthetic rate in leaves that were 1 month old. We measured P sat five times (October 2014, February 2015, May 2015, July 2015, and October 2015), and the measurements were always performed between 09:00 and 11:00. P sat and gs were determined using a portable gas analyzer (LI-6400, LiCor, Lincoln, NE, USA) with an ambient temperature of 28 • C and an ambient CO 2 concentration of 38.0 Pa. The supplemental LED light was 1800 µmol m −2 s −1 photosynthetic photon flux, which was a light saturation level.
After measurement, we collected 0.66 cm 2 of teak leaf disks and stored them at −18 • C. For the extraction of chlorophyll in the leaves, we used dimethyl sulfoxide (DMSO), and determined using a spectrophotometer (AE-VIS721). The concentrations of chlorophyll a and b were calculated according to a previously reported equation [40].

Measurements of Leaf Water Potential and Concentrations of Chlorophyll
Leaf water potential of teak leaves was measured in February 2015 and May 2015 in the mid and late dry season. For the measurement of water potential, same leaves were used to measure the photosynthetic rate. In general, the leaf water potential was lowest in the afternoon because of transpiration and highest during the night after recovery of water [41]. We collected nine teak leaves from each treatment, placed in a plastic bag containing wet filter paper and stored at 6 • C. We determined water potential using a pressure chamber (Model 600, PMS Instrument Co., Albany, OR, USA) at 13:00-14:00 and 05:30-06:00 the next day.

Analysis of Biomass
The dry masses of leaves, stems, and roots were weighed to determine total biomass of the teak seedlings. We collected all of the organs from four teak seedlings in each of the three blocks randomly at the end of November 2015. The other teak seedlings were left so we could examine long-term effects on teak growth. We collected the aboveground organs, divided them into leaves and stems, and measured their fresh masses. We collected the largest leaf from the leaf samples and measured its length and width. Leaf area was calculated using an equation from Tondjo et al. [42] as follows: where A is leaf area, L is leaf length, and Wi is leaf width.
In addition, about 100 g (fresh mass) of leaf and stem samples were collected and their fresh masses were measured. We collected leaves that were second from the top for analysis. These leaf and stem samples were placed in separate envelopes and oven-dried at 70 • C for 3 days. When the sample fresh mass was <100 g, all samples were placed in a single envelope. After drying, the dry mass of each component was determined and the moisture content was calculated. We calculated the dry mass of all leaves and stems based on their moisture content.
We collected root samples from the area of a circle with a radius of 50 cm from the same teak seedlings. The root samples were washed twice with tap water and air-dried for 12 h. After air-drying, the root fresh mass was measured. In addition, about 100 g (fresh mass) of root samples were collected, and their fresh mass was measured. These root samples were placed in envelopes and oven-dried at 70 • C for 3 days. After drying, the root dry mass of each sample was determined, and total root dry mass was calculated from the moisture content value. In addition, we collected 5 g (fresh mass) of fine roots with diameters <2.0 mm. The fine roots were placed in FAA solution, preserved at 4 • C, and used to observe AM colonization.
We then calculated the relative growth rate (RGR [41]) from the July 2014 and November 2015 data.

Analysis of Mycorrhizal Colonization and Element Concentrations in the Plant Organs
A previous experiment revealed that symbiosis with AM fungi helps teak seedlings acquire nutrients from sandy soil [14]. We observed AM colonization in the fine root samples preserved in FAA solution. The root samples were placed in 1 M KOH and cleared by boiling for 1 h. After boiling, the root samples were stained in 0.05% trypan blue in lactic acid and glycerol (1:1) by boiling for 15 min and then de-stained in lactic acid and glycerol (1:1). The root samples were placed on a Petri dish with a film ruled into 5 mm squares. We observed over 200 spots of roots intersected by grid lines at 7 to 45× magnification using a stereomicroscope. AM colonization was calculated by the gridline intersect method, which is the ratio of the number of infected intersections per total number of root intersections [43].
We analyzed the concentrations of N, P, K, Ca, and Mg in the leaves (located second from the top) and fine roots using the dried plant organ samples. The leaves and fine root samples were washed with distilled water and oven-dried at 70 • C for 3 days. After drying, the leaf and root samples were ground into a fine powder using a sample mill (WB-1; Osaka Chemical Co., Osaka, Japan). We used NC analyzer (Sumigraph NC-220F, Sumika Chemical Analysis Service, Tokyo, Japan) for the analysis of concentration of N. The powdered samples were also used for the analysis of other elements, and we digested by the HNO 3 -HCl-H 2 O 2 method [44]. The digested solution was used for the analysis of concentrations of P, K, Ca, and Mg, and determined by ICP analyzer (ICPE-9000, Shimadzu, Kyoto, Japan).

Statistical Analyses
First, we compared all of these parameters among the three blocks of each treatment using one-way ANOVA in Kyplot 5.0 (Kyens Lab. Inc., Tokyo, Japan). There were no significant differences among the three blocks. Thus, we pooled the mean values of parameters for each treatment.
For the soil texture, tree height, diameter, P sat , gs, concentrations of chlorophyll, leaf water potential, survival of seedlings, RGR, leaf area, mycorrhizal colonization, teak dry mass of each organ, and concentration of elements, we used a two-way ANOVA. We compared fertilizer, bentonite and their interaction. For the soil chemical properties, we used a three-way ANOVA, comparing fertilizer, bentonite, two periods of sampling, and their interactions.
To estimate the concentrations of nutrients needed to grow teak, we performed a correlation analysis of the concentration of elements in soils and in the leaves or roots, and the concentration of elements in the leaves or roots and the RGR.

Soil Moisture Content
Average soil moisture content values in the four treatments are shown in Figure 1. Moisture content was low from January to May 2015, whereas moisture content was high during the rainy season (June-September 2014 and June-August 2015). Soil moisture content significantly differed between months (ANOVA, p < 0.001). . Note: Average Data for the CON treatment were from Kayama et al. [15]. The data for the CON treatment in Tables 1-5, Figures 2-5 were also from Kayama et al. [15]. CON: control, FER.: fertilizer, BEN: Bentonite and BEN + FER.: bentonite and fertilizer.
Significant differences were detected in the soil moisture content data with the use of bentonite by unification of all the data (ANOVA, p < 0.001). However, no significant differences were found with the use of bentonite for each month. Soil moisture content was higher in the BEN and BEN + FER treatments during the rainy season. In contrast, little difference was detected in soil moisture content among the four treatments during the dry season. We could not confirm the increase of soil moisture content at surface layer by application of bentonite in dry season.

Soil Properties
Regarding soil texture, the content of sand was about 80% for each treatment (Table 1), and the contents of sand, silt, and clay were no significant difference by use of fertilizer and bentonite (p > 0.05). We examined the changes in pH, CEC, OM, and various nutrients to compare the data between July 2014 and November 2015 ( Table 2). Soil pH showed significant differences between periods (p < 0.001), and its value was increased in November 2015. However, pH trends differed among the four treatments, and there were significant interactions between fertilizer and bentonite, fertilizer and period, and bentonite and period (p < 0.01). The CEC value showed a significant effect of fertilizer or bentonite (p < 0.01). The CEC value was highest in the BEN + FER treatment. The OM concentration showed a significant difference between the two periods (p < 0.001), and its values were decreased in November 2015. The concentration of N showed a significant difference between the use of fertilizer or bentonite and period (p < 0.001). However, N trends differed among four treatments, and there were significant interactions between fertilizer and bentonite, fertilizer and period, and bentonite and period (p < 0.05). The concentration of N was increased in November 2015, except in the case of the BEN + FER treatment.
The concentration of P showed a significant effect of fertilizer or bentonite (p < 0.01). The concentration of P showed a significant interaction between fertilizer and bentonite (p < 0.001), and the value was the highest in the BEN + FER treatment during two periods. The concentration of Ca showed a significant difference with use of fertilizer or bentonite and period (p < 0.01). The concentration of Ca was increased by the use of bentonite in November 2015, and there was a significant interaction between bentonite and period (p < 0.001). The concentration of Mg showed a significant effect of fertilizer or bentonite (p < 0.01), and its value was high with use of fertilizer in November 2015. In contrast, the concentration of Mg in November 2015 was decreased in the treatment with no fertilization, and there was a significant interaction between fertilizer and period (p < 0.01). The concentration of K showed a significant difference with the use of fertilizer and period (p < 0.001). The concentration of K was decreased with the fertilization treatment in November 2015, and there was a significant interaction between fertilizer and period (p < 0.001). The concentration of Na showed a significant difference with the use of fertilizer or bentonite (p < 0.001). The concentration of Na was decreased in the BEN + FET treatment, and increased in the CON treatment. As a result, there were significant interactions between fertilizer and period, and bentonite and period (p < 0.001). Table 2. Texture Chemical properties (pH, CEC, OM, and elements) of soils in the four treatments (mean ± SE, n = 9) sampled in June 2014 and December 2015. Asterisks indicate significant differences by three-factor ANOVA. * = p < 0.05, ** = p < 0.01 and *** = p < 0.001.
Note. Abbreviation written in Statistical analysis was following. Fer. and F: fertilizer, Ben. and B: bentonite, Per. and P: period.

Growth of Teak Seedlings
From October 2014, tree height and diameter were significantly different among the four treatments (Figure 2). Tree height and diameter showed a significant effect of bentonite, and their values were increased by application of bentonite (Table 3, p < 0.001). On the effects of fertilizer, tree height and diameter showed a significantly difference in October 2014 and November 2015 (p < 0.001). Tree height and diameter were increased by use of fertilizer in November 2015, whereas these values were decreased in October 2014.  Table 3. Statistical analysis for tree height, diameter, photosynthetic rate at light saturation (P sat ), stomatal conductance (gs), and concentration of chlorophyll (a + b) in teak seedlings by two-factor ANOVA. Asterisks indicate significant differences. * = p < 0.05, ** = p < 0.01 and *** = p < 0.001.

Parameter
Jul Teak seedlings of fertilization treatment were dead gradually from December 2014. Dead teak seedlings were also confirmed for no fertilization treatment from February 2015.
Based on the data of dead teak seedlings, we calculated survival rate for each block for the four treatments in November 2015 (Table 4). The FER treatments showed high mortality, the survival rate at the end of the experiment was 20.0%. In contrast, BEN treatment showed the highest value of the survival rate (66.7%). The survival rate showed a significant effect of fertilizer, and their values were decreased by application of fertilizer (p < 0.05). Table 4. Survival rate (Nov. 2015), growth rate (RGR), leaf area, and the percentage of AM fungi colonizing teak seedlings grown in the four treatments (mean ± SE, n = 3 for survival rate, and n = 12 for other parameters). Asterisks indicate significant differences by two-factor ANOVA. * = p < 0.05 and *** = p < 0.001.

Parameter Treatment Statistical Analysis
Survival rate (%)

Photosynthetic Rate
The P sat and gs values showed a significant effect of bentonite except in July 2015 (Table 3, p < 0.05). P sat and gs showed their highest values in the BEN and BEN + FER treatments in October 2014, and the values were decreased in February and May 2015 ( Figure 3). In February 2015, the P sat and gs values showed a significant effect of fertilizer (Table 3, p < 0.05). Moreover, P sat in July and October 2015 showed a significant effect of fertilizer (p < 0.05). The values of P sat were low in the FER treatments until May 2015; however, these values increased at the beginning of July 2015 ( Figure 3).
The concentrations of chlorophylls were significantly affected by use of bentonite, except in May 2015 (Table 3, p < 0.05). In these periods, chlorophyll concentrations showed high values with use of bentonite ( Figure 3). The BEN + FER treatment had the highest chlorophyll values during the experimental period in October 2015. In addition, the concentrations of chlorophylls significantly differed with use of fertilizer in February 2015 (Table 3, p < 0.05). In this period, the concentration of chlorophyll was low in the FER treatment ( Figure 3).

Leaf Water Potential
Leaf water potential showed a significant difference with the use of bentonite (p < 0.01), and its value was high in the BEN and BEN + FER treatment (Figure 4). In contrast, the water potentials of the CON treatment in February 2015 and FER treatment in May 2015 were the lowest, and their values were <−1.9 MPa.
The leaf water potential value at predawn showed a significant difference with the use of bentonite (p < 0.001), and its value in the BEN and BEN + FER treatment was increased compared with the value in the afternoon (Figure 4). In contrast, water potential values in CON and FER treatments were <−1.0 MPa in February and May 2015. . Photosynthetic rate at light saturation (P sat ), stomatal conductance (gs), and concentration of chlorophyll (a + b) in teak seedlings grown in the four treatments (9:00-11:00, mean ± SE, n = 9). Note. n = 6 in the FER. and BEN + FER treatments from May 2015 because of dead seedlings.

Biomass and Growth Parameters of Teak Seedlings
The average leaf, stem, and root dry masses at the beginning of the experiment were 1.6, 1.5, and 2.1 g, respectively. The AM colonization value was 21%.
The dry masses of each organ at the end of the experiment were significantly affected by use of fertilizer or bentonite ( Figure 5, p < 0.001). The values of dry masses were markedly increased in the BEN + FER treatment. For leaf and stem dry masses, there was a significant interaction between fertilizer and bentonite (p < 0.05), and their increases were not obvious for the BEN treatment. The dry mass of each organ in the CON treatment was quite small.  The values of RGR and leaf area showed a significant difference with use of fertilizer or bentonite (Table 4, p < 0.001). High values of RGR and leaf area were detected in the BEN + FER treatment. AM colonization showed a significant effect of bentonite (Table 4, p < 0.05), and its value was high in the BEN treatment.

Nutrient Concentrations
The N and P concentrations in leaves and roots showed a significant effect of fertilizer (Table 5, p < 0.01), and these values were increased by fertilization. For the N concentration in roots, the value of the BEN + FER treatment was not high compared with the BEN treatment; as a result, there was a significant interaction between fertilizer and bentonite (p < 0.05). The K concentration in the roots showed a significant effect of fertilizer or bentonite (p < 0.05), and its value was increased by use of bentonite and fertilizer. However, the concentration of K in BEN treatment was not high compared with CON treatment. Therefore, we examined total content of K in roots by multiplying root dry mass and concentration of K. Total content of K in roots was 144 mg for CON treatment, 587 mg for FER treatment, 452 mg for BEN treatment, and 2120 mg for BEN + FER treatment. Content of K in roots showed a significant effect of fertilizer or bentonite (p < 0.01, ANOVA). Table 5. Concentrations of elements (N, P, K, Ca and Mg; dry mass) in leaves and roots of teak seedlings grown in the four treatments (November 2015, mean ± SE, n = 12). Asterisks indicate significant differences by two-factor ANOVA. * = p < 0.05, ** = p < 0.01 and *** = p < 0.001. The Ca concentrations in leaves showed a significant effect of fertilizer (Table 5, p < 0.001), and its value was increased by fertilization. The Mg concentration in the leaves and roots showed a significant effect of bentonite (p < 0.01). The Mg concentration in the leaves and roots was decreased with the use of bentonite.

Element
For the relationship between nutrient concentrations in the soils (sampled in Nov. 2015) and plant organs, positive relationship was observed for the N and P concentrations in leaves, and K concentrations in roots ( Table 6, p < 0.01). For the relationship between RGR and nutrient concentrations in plant organs, a positive relationship was observed between the RGR and P concentration in the leaves ( Table 6, p < 0.001). For the nutrients in roots, a positive relationship was detected between RGR and the K concentration (p < 0.001), whereas a negative relationship was observed between RGR and Ca concentration (p < 0.01). Table 6. Regression coefficients of relationships between concentrations of elements in the leaves or roots of teak seedlings (dependent variable) and concentrations of elements in the soils (independent variable, Nov. 2015, n = 36), and the RGR (dependent variable) and concentrations of elements in the leaves or roots (independent variable) of teak seedlings (n = 48). ** p < 0.01 and *** p < 0.001.

Effects of Fertilizer and Bentonite on Soil
With regard to the effects of application of fertilizer, we confirmed not only an increase in N, P, and K, but also an increase in CEC, Ca, and Mg ( Table 2). The chemical fertilizer used in our experiment probably contained Ca and Mg in the secondary material; as a result, these nutrients were also increased by application of fertilizer. Based on the soil dry mass in a hole (42 kg) and concentrations of Ca and Mg in soil, we quantified the contents of these nutrients in fertilizer. 100 g of fertilizer was contained 1.1 g of Ca and 0.2 g of Mg. Additionally, there is a possibility that fertilizer might have contained material that would increase the CEC value. In the case of bentonite, CEC, Ca, and Mg were increased ( Table 2). Bentonite was contained Na; however, its concentration was decreased at the end of experiment for BEN + FER treatment (Table 2). In contrast, application of bentonite did not change the texture (Table 1).
At the end of the experiment, soil pH was increased by treatment with fertilizer and bentonite ( Table 2). This trend may be attributable to the increase in CEC with use of fertilizer or bentonite. Moreover, the use of bentonite increased the concentration of Ca in the soil (Table 2). In contrast, the FER treatment did not increase the Ca in the soil, and fertilizer had no capacity to increase Ca. Thus, we confirmed that application of bentonite alone can increase Ca in the soil. We also examined the retentive capacity of the bentonite with regard to the nutrients in fertilizer. Concentration of P in soil was maintained for the BEN + FER treatment until November 2015, despite the lack of increase in the BEN treatment (Table 2). In contrast, the concentrations of N and K in the soil were decreased for the BEN + FER treatment in November 2015 (Table 2). Thus, bentonite has a retentive capacity for P in fertilizer.
The trait to increase CEC by application of fertilizer is originated from the increase of soil colloid retention by application of K ions [45]. According to Bhatt [46], there have been cases where the use of fertilizer increased CEC and concentrations of Ca and Mg in soil. In the case of bentonite, Mg was increased by application of bentonite, whereas Ca was not increased [14]. Therefore, the increase of Ca by application of bentonite is probably concerned by long period of soil improvement. Noble et al. [26] and Soda et al. [47] reported that application of bentonite increased soluble inorganic sources of cations; as a result, pH and concentrations of various cations were increased in the soil. On the mechanism to increase cations in soil, sodium bentonite has a characteristic to bind Ca 2+ ion from pore water in natural soil, and exchange Na + ion in the double layer of bentonite [48]. The exchanged Ca 2+ ion at the site of landfill was increased for two years by use of geosynthetic clay liners, which contained sodium bentonite [48]. This exchanging is enhanced by drywet-cycles, and evaporation of water cause the ion concentration in pore water to rise [48]. Thus, application of bentonite may be able to bind Ca from the pore water in sandy soil, as a result, the concentration of Ca in the soil may be increased at the end of experiment. The increase of Ca in soil by application of sodium bentonite may be a peculiar characteristic at the region of the climate with dry season.
Moreover, sodium bentonite is prepared by exchanging of polymeric metal species such as aluminum [Al] [49]. Modified bentonite consists chemical complex of Al 3+ , and called Al pillared bentonite. The Al 3+ in Al pillared bentonite can bind anion of phosphate [49], as a result, P in fertilizer probably retain by application of bentonite. The effects of the increase of Al was little by application of bentonite [14]. Bentonite is utilized as a slow-release fertilizer material [29,50]. Concurrent use of bentonite and fertilizer may play a similar role to that of slow-release fertilizer.

Effects of Fertilizer and Bentonite on Teak Biomass
The teak seedlings in the BEN + FER treatment had the largest tree height, diameter and dry masses for each organ and the largest leaf area values at the end of the experiment (Figures 2 and 5 and Table 3). Application of bentonite showed positive effect for increase of tree height and diameter of teak seedlings from October 2014 ( Figure 2, Table 3). In contrast, the effects of fertilizer for the growth of teak was late compared with bentonite, and growth acceleration by fertilizer was shown after July 2015 ( Figure 2, Table 3). Thus, the use of bentonite resulted in accelerated growth of teak seedlings in sandy soil, and effects of fertilizer is appeared after one year. On the effects of nutrients for the application of bentonite, only K in the roots showed a significant increase in concentration and total content with use of bentonite (Table 5). Thus, bentonite had little capacity to increase the concentration of nutrients in the plant organs.
Croker et al. [51] reported an obvious increase in the concentration of K in plant organs with application of bentonite, and this trend was attributed to its migration by diffusion when there was high soil water content. We confirmed an increase in soil moisture in the BEN and BEN + FER treatments during the rainy season ( Figure 1). Thus, the increase in uptake of K was probably attributable to migration by diffusion under the high soil water content of the BEN treatment.
To estimate total nutrient uptakes, we examined the total content of each nutrient, which was calculated using the concentration of nutrients and dry mass of leaves or roots. The total content of each nutrient in the leaves and roots showed markedly high values in the BEN + FER treatment and a significant difference with use of fertilizer or bentonite (data not shown, p < 0.05 ANOVA). Thus, large amounts of nutrients were absorbed from the soil in the BEN + FER treatment, which accelerated teak seedling growth. Several reports have indicated that plant production by concurrent use of bentonite and fertilizer was larger than that by single use of fertilizer [24,31]. Thus, the use of bentonite contributes to efficient uptake of the fertilizer.
The teak seedlings in the BEN treatment showed significant high values of height and diameter from October 2014 (Figure 2), and significant large dry masses for each organ at the end of experiment ( Figure 5). However, the values of dry masses in the BEN treatment were much smaller than those in BEN + FER treatment ( Figure 5). Thus, growth of teak seedlings in the BEN treatment is probably suppressed by nutrient deficiency.
We examined nutrient deficiency in the leaves according to the values of Zech and Drechsel [52]. The concentrations of N, P and Ca for visible symptoms of deficiency were below 12.0 mg g −1 , 1.0 mg g −1 , and 5.5 mg g −1 , respectively [52]. We did not confirm a deficiency of N in leaves in each treatment (Table 5). We also confirmed that more than 80% of the teak seedlings in each treatment were colonized by AM fungi (Table 4). Numerous studies have reported that AM fungi play an important role acquiring N [53]. Raghu et al. [32] confirmed that teak seedlings were increased the uptake of N after inoculation of AM fungi. Thus, indigenous AM fungi distributed in the sandy soil environment might have infected the roots of teak seedlings, which could have enhanced N uptake. Moreover, Ca in the leaves did not show a deficiency in the BEN and BEN + FER treatments (Table 5). In contrast, a deficiency in P was shown for the CON and BEN treatments (Table 5). Therefore, the limiting factor to growth suppression of BEN treatment is probably P deficiency in leaves. Similar results were shown in a pot experiment by single use of bentonite [14], and growth acceleration was not confirmed. Fertilization with P is essential to prevent a deficiency in teak seedlings.
We also verified the most effective nutrients for growth acceleration of teak seedlings. Based on the correlation coefficient, the RGR showed a significant positive correlation with K in the roots and P in the leaves (Table 6). K in the roots and P in the roots were also associated from these concentrations in the soils (Table 6). Thus, the teak seedlings grown on the habitats of high concentrations of K and P in soils can accumulate K in the roots and P in the leaves; as a result, teak seedlings are accelerated their growth.
Behling et al. [54] reported the nutrient dynamics in teak roots. Teak has a characteristic low efficiency of K uptake, and teak demands a greater amount of K in the roots [54]. Chanan et al. [55] also reported a relationship between growth and nutrients in leaves, and P is a macronutrient that limits teak growth. Thus, the level of accumulation of K in roots and P in leaves likely affects the growth of teak.
On the other hand, many teak seedlings of fertilizer treatment were died from December 2014, and the survival rate in November 2015 was significantly decreased (Table 4). Previous experiments reported that the use of fertilizer decreased the survival of seedlings of woody species [56]. When a seedling is exposed high salt concentration around the roots, dehydration occurs [56]. Especially, low CEC soil, such as sandy soil of this experiment can be associated with serious damage to plants by fertilizer [57]. Thus, fertilization treatments may enhance the damage caused by fertilizer.

Effects of Fertilizer and Bentonite on Physiological Characteristics
High P sat values were observed in the teak seedlings from the BEN treatment in October 2014 and February 2015 ( Figure 3). In addition, a high P sat value was observed in the BEN + FER treatment during October 2015 ( Figure 3). In contrast, the N and P concentrations, which were positively associated with the photosynthetic rate [41,58,59], did not show high concentrations (Table 5). However, the chlorophyll concentrations were high at the same time ( Figure 3). Chlorophyll concentrations are closely related to photosynthetic rate [60,61]. Thus, the photosynthetic rate in teak leaves is regulated by the concentration of chlorophyll in the leaves.
Moreover, high leaf water potential values at predawn were detected in the BEN and BEN + FER treatments in the dry season ( Figure 4). Thus, teak seedlings treated with bentonite probably took up water from deep soil layer during the night, so the predawn leaf water potential was high. In addition, AM colonization was significantly increased by application of bentonite (Table 4).
Nemec [62] reported that the use of bentonite enhanced the development of hyphae of AM fungi. This trait is probably concerned the increase of Ca in soil by application of bentonite. Several reports showed the increase of the colonization of AM fungi by application of Ca [63,64]. In addition, the development of hyphae of AM fungi enhances water uptake and increases drought stress tolerance [65]. Thus, teak seedlings treated with bentonite can enhance water uptake by the development of hyphae of AM fungi. As a result, bentonite mitigated drought stress in the teak seedlings during the dry season. A similar trend was confirmed by our preliminary experiment [14] and a crop experiment [66].
In contrast, low P sat , gs, and chlorophyll concentrations were observed in the FER treatment during October 2014 and February 2015 (Figure 3). February 2015 was the middle of dry season, and leaves suffered from drought stress owing to low leaf water potential at predawn (Figure 4). Suppression of these parameters in the FER treatment is probably attributable to drought stress. In contrast, October 2014 was not in the dry season; however, the P sat , gs, and chlorophyll concentrations showed low values. We could not explain this result by climatic conditions, and we analyzed the concentration of nutrients in the leaf during the same period. The concentration of N in the CON treatment in October 2014 was 34.0 mg g −1 .
The concentration of N in teak leaves does not typically exceed 30 mg g −1 in the wild [52,67], and this concentration is probably considered an excessive level. One study reported that the concentration of chlorophyll, photosynthetic rate, and stomatal conductance decrease in woody species raised with over 30 mg g −1 N in the leaves [68]. Thus, excess N might have suppressed various physiological parameters in the FER treatment.

Conclusions
Based on a preliminary pot experiment, we concluded that bentonite would be useful for improving water content of sandy soil. In contrast, the growth of teak seedlings of pot experiment was not accelerated because of few nutrients in bentonite. Based on previous results, we curried out a verification test with fertilizer and bentonite by planting teak seedlings in a field environment, and monitored early growth period. We confirmed that bentonite had high retentivity of P in fertilizer, and teak seedlings absorbed nutrients efficiently with the application of bentonite and fertilizer. We also confirmed the accumulation of K in roots with the application of bentonite. Moreover, the soil Ca concentration increased during the experimental period, and teak did not show a Ca deficiency without application of Ca. Water availability was increased, and drought stress in the teak seedlings was mitigated by the application of bentonite. Based on these positive effects on the concurrent use of bentonite and fertilizer, teak seedlings showed markedly growth acceleration. In contrast, a single use of bentonite did not clearly accelerate the growth of teak because of the effect of P deficiency. The single use of fertilizer increased biomass and nutrients in the plant organs; however, the photosynthetic rate was decreased and drought stress ensued. As a result, the use of fertilizer alone showed high mortality, and did not promote marked growth acceleration.
We concluded that the concurrent use of fertilizer and bentonite showed a synergistic effect for the growth of teak seedlings. This method was useful for accelerating the growth of teak seedlings in sandy soil in northeast Thailand. In particular, we confirmed the beneficial effects of bentonite, which has not been widely studied for its application to silviculture. In contrast, we also confirmed that fertilization of teak seedlings was better after first dry season. In the future, we intend to examine the quantitative effects of bentonite to establish silviculture methods.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to policy of the institute.