text:
Morphological Features and Biomass Partitioning of Lucerne Plants ( Medicago sativa L.) Subjected to Water Stress

: Drought is one of the major abiotic stresses a ﬀ ecting the morphological, physiological, and metabolic processes of plants, and hence their growth and production on a global scale. Lucerne ( Medicago sativa L.) is one of the most popular pasture species in semi-arid regions and plays a critical role in sustaining agricultural systems in many areas of the world. In order to evaluate the e ﬀ ect of water deﬁcits on the growth and biomass distribution in di ﬀ erent tissues of lucerne, plant height, leaf dry weight, leaf number and area, root dry weight, taproot length and lateral root number, and stem dry weight were measured at four stages from the seedling to ﬂowering stages under three water regimes: (i) adequate water supply (minimum soil water content 85% pot capacity (PC)), (ii) moderate water stress (65% PC), and (iii) severe water stress (45% PC), imposed under a rainout shelter. With limited water supply, plant height, leaf number, leaf area and dry weight, taproot length, and total biomass were reduced, while lateral root numbers increased. The number of smaller leaves and root dry weight increased under moderate water stress, whereas severe water stress reduced them. Leaf, stem, and total dry weight were all reduced by the water deﬁcits, but leaf dry weight was reduced the most and root dry weight the least, so there was a redistribution of biomass towards the roots, increasing the root–shoot ratio. These results help us to understand the response of lucerce to water stress and assist in developing a foundation for the sustainable use of lucerne in semi-arid agricultural systems.

Introduction
Drought stress is one of the most important abiotic stresses on a global scale, causing major crop losses [1]. Adverse growing conditions, especially an increase in aridity, are the main reasons for reductions in the potential yield of major crops [2]. The tolerance of plants to drought stress is a complex phenomenon that involves specific morphological, physiological, biochemical, and metabolic adaptations that affect plant growth, development, and survival [3][4][5][6]. Plants respond to water deficits by strategies including drought escape, drought avoidance, and drought tolerance [7,8], but the adjustment of phenology to escape or avoid severe water shortage and the metering of water use to provide sufficient resources for the reproductive phase predominate in grain crops [9]. For perennial pasture species such as lucerne, the ability to use available water to maximize aboveground biomass, and survival of drought episodes, are more important than conserving water for the reproductive phase [10]. Nasreddine et al. [11] reported that the growth patterns of different plant organs are affected differently by drought stress. For example, plant morphological components such as leaf area will often be modified to reduce water loss by transpiration [12], and the distribution of assimilates are altered to maximize root exploration of the soil, leading to an increase in the root-shoot dry matter ratio (R/S) [13]. Pasture yield is also influenced by the redistribution of stored assimilates into economically important organs [14].
Lucerne/alfalfa (Medicago sativa L.) is one of the most popular pasture species in semi-arid regions [14,15] and plays a critical role in sustaining agricultural systems by diversifying crop rotations, providing highly nutritious livestock feed, enhancing soil quality through biological nitrogen fixation [16][17][18], and benefiting soil and water conservation [19]. Lucerne is an extremely adaptable plant that can be grown under a wide range of climatic conditions from the equatorial to polar regions [20,21]. However, water availability is one of the primary factors limiting its distribution, growth, and development. The planted area of lucerne is the largest among forage species in China. It has been grown for at least 150 years on the Loess Plateau of northwestern China [22] where the long-term annual precipitation is 250-400 mm [23] and where precipitation is the major source of water supply for crops and pastures. M. sativa L. cv. Xinjiangdaye was successfully introduced to the Loess Plateau in 1989, but little research has been undertaken on why it has been such a successful pasture species for the past three decades. There is therefore a need to identify the morphological and physiological features of lucerne that influence its growth and productivity in conditions of water limitation. Although the general effects of drought on growth of lucerne have been studied [24][25][26], and an expanded root system reported under water-limited conditions [27], further investigation on the growth of lucerne under water-limited conditions at the early growth stage is warranted, as lucerne is reportedly not drought-tolerant during the first 2 to 5 months of its growth [26]. In particular, growth and physiological parameters such as the partitioning of biomass to different tissues/organs need to be clarified and quantified.
The present study was initiated to investigate the effects of drought stress on vegetative growth, leaf features, and biomass distribution over the early growth stages of M. sativa Xinjiangdaye, from seedling emergence to flowering, in order to understand the morphological and physiological basis of lucerne establishment, growth, and development under water-limited conditions and the likely limitations to its early growth in semi-arid environments.

Materials
Lucerne (Medicago sativa. L. cv. Xinjiangdaye) seeds were kindly provided by the Key Laboratory of Grassland Ecosystems of the Ministry of Education of China. Plastic pots, 350 mm in diameter and 400 mm in height with holes at the base to allow for drainage, were used to grow the plants. Each pot was filled with 9.5 kg of air-dried sandy-loam topsoil collected from the campus of Gansu Agricultural University with a measured pot capacity (PC) of 25.7% (determined by watering to complete saturation, allowed to drain for 24 h before weighing (wet weight), and oven-drying the soil for 24 h (dry weight), then PC = ((wet weight − dry weight)/(dry weight − empty pot weight)) × 100). The soil in each pot was mixed with 0.5 kg of livestock manure and 1 L of nutrient solution (NH 4 NO 3 : 5.42 g/L, KH 2 PO 4 : 2.33 g/L) was applied before sowing.

Methodology
A total of 48 pots (three water treatments, plants harvested at four growth stages, four replicates) were arranged in a randomized complete block design, with water treatments and harvest stages completely randomized in four replicate blocks. The three water treatments were (i) adequate water supply (WW, minimum soil water content (SWC) = 85% PC); (ii) moderate water stress (MS, minimum SWC = 65% PC), and (iii) severe water stress (SS, minimum SWC = 45% PC). The experiment was conducted in 2017 under a rainout shelter (3 m long × 5 m wide × 2.7 m high that was open except Agronomy 2020, 10, 322 3 of 10 during rain events) in the experimental field of the College of Forestry at Gansu Agricultural University (36 • 09' N, 103 • 70' E, altitude 1620 m), Lanzhou, Gansu Province, China. Before sowing, each pot was watered until water drained from the holes at the base of the pots. Twenty seeds were sown in each pot on 1 April 2017. A preliminary experiment showed that from 7 days after sowing (DAS), no further lucerne seeds were likely to emerge, so on 8 April, plants were thinned to three per pot and the three water treatments imposed. In all treatments, the SWC reached the predetermined levels of 85%, 65%, and 45% PC within 48 h. Each pot was weighed at 17.30 h Beijing Standard Time (BST) each day and re-watered to the three predetermined levels of 85%, 65%, and 45% PC. After 14 days (22 April-the seedling stage at which the plants had two leaves), 28 days (6 May-branching stage at which the internodes were elongated with branches on the main stem), 42 days (20 May-squaring when many buds developed), and 56 days (3 June-flowering when many flowers had opened), the three plants per pot in each replicate were harvested at soil level and the mean values of the three plants were used for further analysis. Plant height and leaf area were measured with a ruler and leaf area meter (Li-3100, LiCor, Lincoln, NE, USA), respectively, and leaf number was counted. The roots were washed with water to remove the soil, wiped dry with a paper towel, the length of the taproot was measured, and the lateral root number was counted. The harvested plants were separated into leaves, stems, and roots, and the dry weight of each was obtained after oven-drying at 65 • C for 48 h. The growth rate (GR) and root-to-shoot dry weight ratio (R/S) were calculated. GR = (dry weight at t 2 − dry weight at t 1 )/(t 2 − t 1 ) (1) in which t 1 and t 2 are the days to the different growth stages.
R/S = root dry weight/(stem dry weight + leaf dry weight)

Statistical Analyses
The general analysis of variance (ANOVA) in SPSS 12.0 software (SPSS, Chicago, IL, USA) was used to test for significance of all measured variables. Before each ANOVA, the homogeneity of the variances was tested and the data met the normal and equal variance assumptions of ANOVA without transformation. Comparisons of means among organs and between treatments were carried out via Tukey's test at p < 0.05.

Effect of Water Treatments on Plant Height and Leaf Growth
In the water-sufficient treatment (85% PC), the height of the plants at the seedling stage was 157 ± 5 mm, and by flowering this had reached 541 ± 22 mm ( Figure 1A). Height growth was not linear over this period, but plants grew at 7.8 ± 2.1 mm day −1 between seedling and branching, 6.9 ± 0.9 mm day −1 between branching and squaring, and 5.9 ± 0.5 mm day −1 between squaring and flowering. In the moderate water stress treatment (65% PC), height was reduced at all stages compared with the water-sufficient treatment ( Figure 1A) due to a slowing of the rate of height growth between the seedling and squaring stages. In the severe stress treatment (45% PC), the height of the plants was reduced further at all stages ( Figure 1A); the rate of growth was approximately linear at 4.5 ± 0.3 mm day −1 from the seedling to flowering stages.
the leaf size was reduced by about 20% by the moderate stress (65% PC), and by about 35% by the severe stress treatment (45% PC) ( Figure 1D).

Figure 1.
Effect of watering treatments (WW: water-sufficient (85% pot capacity (PC)), MS: moderate water stress (65% PC), and SS: severe water stress (45% PC)) on (A) plant height, (B) leaf area per plant, (C) leaf number per plant, and (D) leaf area per leaf at the four growth stages: seedling, branching, squaring, and flowering. The bars are ± one standard error of the mean (n = 4). Different lower case letters represent significant differences at p < 0.05 according to Tukey's multiple range test．

Effect of Water Treatments on Taproot Length and Lateral Root Number
By the seedling stage, the length of the taproot in the water-sufficient treatment (85% PC) was 183 ± 15 mm, and this increased to 387 ± 23 mm by branching and 628 ± 35 mm by flowering ( Figure  2A); i.e., the root length growth was 16% greater than the plant height growth over the same period ( Figure 1A). The rate of length growth was fastest between the seedling and branching stages at 14.5 mm day -1 , and decreased to about 60% of this rate between branching and flowering. Taproot length was reduced by both water stress treatments ( Figure 2A); at the seedling and branching stages, the taproot was not significantly shorter in the moderate stress treatment (65% PC, p < 0.05), but at flowering it was significantly reduced (p < 0.05) by 19%, compared with the water-sufficient lucerne ( Figure 2A). The severe stress treatment (45% PC) reduced the rate of taproot growth by 50% in the period between the seedling and branching stages and to a more severe degree thereafter, so that by flowering the taproot was 325 ± 12 mm long in the severe stress treatment, 478 ± 25 mm long in the moderate stress treatment, and 628 ± 35 mm long in the water-sufficient plants ( Figure 2A).
Water stress increased the number of lateral roots in the lucerne ( Figure 2B). The lateral root number in the water-sufficient treatment increased approximately linearly from 7 plant -1 at the seeding stage to 25 plant -1 at flowering. Moderate and severe stress increased the number of lateral roots by 12%-18% and by 25%-29%, respectively, over the water-sufficient treatment. While the numbers were always higher in the severe than the moderate stress at all stages of development, the differences were only significant (p < 0.05) at the branching stage. (Figure 2B). In the water-sufficient treatment (85% PC), the leaf area plant −1 increased approximately linearly from 0.11 ± 0.03 m 2 plant −1 at the seedling stage to 1.14 ± 0.37 m 2 plant −1 at flowering ( Figure 1B). In the moderate stress treatment (65% PC), the leaf area was reduced by about 20% at all stages, but this was only statistically significant at the flowering stage ( Figure 1B, p < 0.05). However, the leaf area was significantly reduced in the severe water stress treatment (45% PC) by about 60% at all stages ( Figure 1B, p < 0.05). The reduction in area was the result of a reduction in both the number of leaves ( Figure 1C) and area per leaf ( Figure 1D). The moderate stress (65% PC) only had a significant effect on leaf number at the seedling stage of growth (p < 0.05), but the severe stress treatment (45% PC) reduced the number of leaves by about a third at all stages of development ( Figure 1C), while the area per leaf was reduced by the moderate stress ( Figure 1D). Throughout the period of development, the leaf size was reduced by about 20% by the moderate stress (65% PC), and by about 35% by the severe stress treatment (45% PC) ( Figure 1D).

Effect of Water Treatments on Taproot Length and Lateral Root Number
By the seedling stage, the length of the taproot in the water-sufficient treatment (85% PC) was 183 ± 15 mm, and this increased to 387 ± 23 mm by branching and 628 ± 35 mm by flowering ( Figure 2A); i.e., the root length growth was 16% greater than the plant height growth over the same period ( Figure 1A). The rate of length growth was fastest between the seedling and branching stages at 14.5 mm day −1 , and decreased to about 60% of this rate between branching and flowering. Taproot length was reduced by both water stress treatments ( Figure 2A); at the seedling and branching stages, the taproot was not significantly shorter in the moderate stress treatment (65% PC, p < 0.05), but at flowering it was significantly reduced (p < 0.05) by 19%, compared with the water-sufficient lucerne ( Figure 2A). The severe stress treatment (45% PC) reduced the rate of taproot growth by 50% in the period between the seedling and branching stages and to a more severe degree thereafter, so that by flowering the taproot was 325 ± 12 mm long in the severe stress treatment, 478 ± 25 mm long in the moderate stress treatment, and 628 ± 35 mm long in the water-sufficient plants (Figure 2A). . Different lowercase letters represent significant differences at P < 0.05 according to Tukey's multiple range test.

Effect of Water Treatments on Leaf, Stem, and Root Dry Weight
The total dry weight at the seedling stage in all three water treatments was 0.14 ± 0.08 g plant −1 , and increased in the water-sufficient plants to 0.51 ± 0.11 g plant −1 by flowering ( Figure 3A). With reduced water supply, the total dry weight accumulation decreased in both the moderate water stress and severe water stress treatments, so that by flowering the total dry weight in moderate water stress (65% PC) and severe water stress (45% PC) was 93% and 67% of that in the water-sufficient treatment, respectively ( Figure 3A). The reduction in total dry weight in the moderate stress treatment was always less than 10% and was not statistically significant (p > 0.05) between the seedling and squaring stages.
Both the leaf and stem dry weights were reduced by the water stress treatments ( Figure 3B, C). In the water-sufficient treatment (85% PC), the leaf dry weight (0.05 ± 0.01 to 0.20 ± 0.06 g plant −1 ) was similar to the stem dry weight (0.05 ± 0.01 to 0.18 ± 0.03 g plant −1 ), but the reduction in dry weight was greater in the leaf than in the stem at all developmental stages, particularly at squaring and flowering ( Figure 3B, C). For example, at flowering, the reduction in leaf dry weight was 26% and 56% in the moderate and severe stress treatments, respectively, while in the stems the reduction in dry weight was 6% and 31%, respectively.
In the moderate water stress treatment, the root dry weight was always higher than in the watersufficient treatment, significantly so at squaring and flowering ( Figure 3D, p < 0.05). By contrast, the root dry weight in the severe stress treatment was significantly lower (p < 0.05) than in the watersufficient and moderately stressed treatments at branching, squaring, and flowering ( Figure 3D). At flowering, the root dry weight was 16% higher in the moderate stress treatment and 10% lower in the severe stress treatment than in the water-sufficient treatment. Water stress increased the number of lateral roots in the lucerne ( Figure 2B). The lateral root number in the water-sufficient treatment increased approximately linearly from 7 plant −1 at the seeding stage to 25 plant −1 at flowering. Moderate and severe stress increased the number of lateral roots by 12-18% and by 25-29%, respectively, over the water-sufficient treatment. While the numbers were always higher in the severe than the moderate stress at all stages of development, the differences were only significant (p < 0.05) at the branching stage. (Figure 2B).

Effect of Water Treatments on Leaf, Stem, and Root Dry Weight
The total dry weight at the seedling stage in all three water treatments was 0.14 ± 0.08 g plant −1 , and increased in the water-sufficient plants to 0.51 ± 0.11 g plant −1 by flowering ( Figure 3A). With reduced water supply, the total dry weight accumulation decreased in both the moderate water stress and severe water stress treatments, so that by flowering the total dry weight in moderate water stress (65% PC) and severe water stress (45% PC) was 93% and 67% of that in the water-sufficient treatment, respectively ( Figure 3A). The reduction in total dry weight in the moderate stress treatment was always less than 10% and was not statistically significant (p > 0.05) between the seedling and squaring stages.
Both the leaf and stem dry weights were reduced by the water stress treatments ( Figure 3B,C). In the water-sufficient treatment (85% PC), the leaf dry weight (0.05 ± 0.01 to 0.20 ± 0.06 g plant −1 ) was similar to the stem dry weight (0.05 ± 0.01 to 0.18 ± 0.03 g plant −1 ), but the reduction in dry weight was greater in the leaf than in the stem at all developmental stages, particularly at squaring and flowering ( Figure 3B,C). For example, at flowering, the reduction in leaf dry weight was 26% and 56% in the moderate and severe stress treatments, respectively, while in the stems the reduction in dry weight was 6% and 31%, respectively.
In the moderate water stress treatment, the root dry weight was always higher than in the water-sufficient treatment, significantly so at squaring and flowering ( Figure 3D, p < 0.05). By contrast, the root dry weight in the severe stress treatment was significantly lower (p < 0.05) than in the water-sufficient and moderately stressed treatments at branching, squaring, and flowering ( Figure 3D). At flowering, the root dry weight was 16% higher in the moderate stress treatment and 10% lower in the severe stress treatment than in the water-sufficient treatment. Agronomy 2019, 9, x FOR PEER REVIEW 6 of 10 Dry matter was distributed to the different organs at different rates depending on water availability (Figure 4). At the seedling stage, the distribution of dry matter was similar in the leaves, stems, and roots in all the water treatments, and the R/S ratio was 0.47 (Table 1). In the watersufficient treatment, the R/S ratio was similar at branching to that at seeding, but then decreased at the squaring stage and decreased further at the flowering stage as a greater proportion of the assimilates was distributed to the leaves (Figure 4). With increasing water stress, the total dry matter of the plants decreased and a greater proportion of this dry matter was distributed to the roots and less to the leaves (Figure 4)   Figure 4. Effect of watering treatment (WW: water-sufficient (85% PC), MS: moderate water stress (65% PC), and SS: severe water stress (45% PC)) on the proportion of the total dry matter in the leaves, stems, and roots at the different growth stages: seedling, branching, squaring, and flowering. The values above the bars give the total dry matter (mg/plant) shown in Figure 3A.

Discussion
Water deficits are well known to reduce the growth of most crops and pastures [3,8,10,25,26]. We chose to study the effect of water shortage on the early growth of Lucerne, as it has been stated that "seedling alfalfa (alfalfa during its first 2 to 5 months of growth) is not as drought tolerant as is established alfalfa (lucerne), so moisture stress at this time should be avoided. If soil moisture is Dry matter was distributed to the different organs at different rates depending on water availability (Figure 4). At the seedling stage, the distribution of dry matter was similar in the leaves, stems, and roots in all the water treatments, and the R/S ratio was 0.47 (Table 1). In the water-sufficient treatment, the R/S ratio was similar at branching to that at seeding, but then decreased at the squaring stage and decreased further at the flowering stage as a greater proportion of the assimilates was distributed to the leaves (Figure 4). With increasing water stress, the total dry matter of the plants decreased and a greater proportion of this dry matter was distributed to the roots and less to the leaves ( Figure 4) Agronomy 2019, 9, x FOR PEER REVIEW 6 of 10 Dry matter was distributed to the different organs at different rates depending on water availability (Figure 4). At the seedling stage, the distribution of dry matter was similar in the leaves, stems, and roots in all the water treatments, and the R/S ratio was 0.47 (Table 1). In the watersufficient treatment, the R/S ratio was similar at branching to that at seeding, but then decreased at the squaring stage and decreased further at the flowering stage as a greater proportion of the assimilates was distributed to the leaves (Figure 4). With increasing water stress, the total dry matter of the plants decreased and a greater proportion of this dry matter was distributed to the roots and less to the leaves (Figure 4)   Figure 4. Effect of watering treatment (WW: water-sufficient (85% PC), MS: moderate water stress (65% PC), and SS: severe water stress (45% PC)) on the proportion of the total dry matter in the leaves, stems, and roots at the different growth stages: seedling, branching, squaring, and flowering. The values above the bars give the total dry matter (mg/plant) shown in Figure 3A.

Discussion
Water deficits are well known to reduce the growth of most crops and pastures [3,8,10,25,26]. We chose to study the effect of water shortage on the early growth of Lucerne, as it has been stated that "seedling alfalfa (alfalfa during its first 2 to 5 months of growth) is not as drought tolerant as is established alfalfa (lucerne), so moisture stress at this time should be avoided. If soil moisture is . Effect of watering treatment (WW: water-sufficient (85% PC), MS: moderate water stress (65% PC), and SS: severe water stress (45% PC)) on the proportion of the total dry matter in the leaves, stems, and roots at the different growth stages: seedling, branching, squaring, and flowering. The values above the bars give the total dry matter (mg/plant) shown in Figure 3A. Table 1. Effect of three watering treatments (WW: water-sufficient (85% PC), MS: moderate water stress (65% PC), and SS: severe water stress (45% PC)) on the growth rate and root/shoot ratio of lucerne at different growth stages: seedling, branching, squaring, and flowering. The mean growth rates and root/shoot ratios for the period from the beginning of the water treatments to flowering are also given. The values are means ± standard error of the mean (n = 4).

Discussion
Water deficits are well known to reduce the growth of most crops and pastures [3,8,10,25,26]. We chose to study the effect of water shortage on the early growth of Lucerne, as it has been stated that "seedling alfalfa (alfalfa during its first 2 to 5 months of growth) is not as drought tolerant as is established alfalfa (lucerne), so moisture stress at this time should be avoided. If soil moisture is inadequate during the establishment phase, excessive plant mortality and stand loss can occur" [26]. However, in arid and semi-arid areas in which lucerne is grown, drought stress cannot always be avoided during establishment and early vegetative growth. In this study, we showed that water deficits in the vegetative stage reduced the production of aboveground forage, largely as a result of a reduction in leaf biomass and leaf area due to the production of smaller leaves, particularly under severe stress (45% PC). The water stress treatments also reduced the length of the taproot, but increased lateral root development and, in the moderate stress (65% PC) treatment, increased the root biomass. The increase in dry weight of the roots in the moderate and severe water stress treatments with development was proportionally greater than in the leaves, resulting in the R/S ratio increasing with increasing water deficit.
The development of leaf area is important in determining the growth rate and water use of lucerne, and it increased linearly in the water-sufficient plants. The lucerne plants were able to maintain their leaf area and the number of leaves until flowering at moderate stress (65% PC), but severe stress (45% PC) significantly reduced the leaf area throughout growth from the seedling to the flowering stages as a result of fewer (42% fewer by flowering) and smaller (40% smaller at flowering) leaves compared with leaves in the water-sufficient plants. Maintenance of leaf area at moderate stress with an increased number of smaller leaves will maintain photosynthesis and biomass accumulation, and hence forage amount and quality, but at the expense of greater water use. We did not measure water use or photosynthesis in this study, but the rate of total biomass accumulation was similar in the water-sufficient lucerne and moderately stressed lucerne until flowering, suggesting that maintenance of leaf area was an adaptation to limited water in lucerne. By contrast, leaf area was markedly reduced by the severe water stress treatment, indicating that the reduction in leaf area was an adaptation to the very limited water availability. These morphological responses are associated with plant drought adaptation strategies, and plasticity of leaf number and size is an important component of drought adaptation [28].
The changes in total dry weight from the 5-day-old seedlings when the treatments were imposed to flowering showed that the overall growth rate decreased from 9.2 mg day −1 in the water-sufficient lucerne plants to 8.3 mg day −1 (91% of the water-sufficient treatment) in those subjected to moderate water stress, and to 6.0 mg day −1 (66% of the water-sufficient treatment) in those in the severe stress treatment. In all three water treatments, the fastest growth occurred at the seedling and branching stages, the slowest at the flowering stage ( Table 1). The more severe the water stress, the greater the proportion of seedling and branching stage growth in terms of the entire growth from the seedling to flowering stages ( Table 1). The R/S ratio remained relatively constant (0.42 to 0.50) in the moderate stress treatment (Table 1). However, in the severe stress treatment, the assimilate distribution to the leaves was severely reduced by the water stress while assimilate distribution to the roots was similar to that in the water-sufficient lucerne, so the R/S ratio increased to 0.55 at branching, reached 0.73 at squaring and then decreased again to 0.38 at flowering (Table 1), primarily as a result of a smaller proportion of dry matter being distributed to the leaves (Figure 4). Not only was root dry weight increased in the moderate stress treatment, but also the number of lateral roots, enabling greater water uptake from the drying soil. This confirmed a previous observation that roots of lucerne seedlings are more resilient to water deficits than the leaves or stems, and not just at the seedling stage [29], but also throughout the vegetative stage to flowering. In this way, plants can reduce the consumption of water by minimizing the evaporative area and water use, a drought-avoidance strategy [30], while fully exploring the soil volume for water. Similar results have been reported [31] in which moderate and severe drought stress reduced the growth of shoots rather than roots, compared to the water-sufficient treatments. Our results were in accordance with these studies: leaf area and taproot length decreased in response to water stress, which can be explained by cessation of cell division and elongation [32]. Rasekh et al. indicated that water deficit conditions during vegetative growth influence cell division and enlargement, which consequently suppress plant height, leaf growth, and its development [33]. However, the reduction in taproot growth may have been the result of the restricted root growth in pots, particularly as a result of frequent watering from the top of the pot [34] The different water stress levels applied in our experiment induced different growth responses in lucerne, indicating that the severity of water stress and the development of water stress must be taken into consideration when developing a management strategy for lucerne. The observation that taproot length was reduced by the reduction in water availability suggests that if irrigation is available, application of water to prevent a reduction in taproot growth would be beneficial, as anecdotal evidence suggests that the root system should be 1 to 1.5 m depth in the soil before the onset of severe stress [26]. If irrigation is not available, withholding grazing or harvesting in the late vegetative stage should be avoided to maintain biomass and reserves for regrowth when the water shortage is relieved.


extfieldsofstudy: Medicine Chemistry

provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2020.jsonl.gz.gz

text:
Effects of Combined α-Amylase and Endo-Xylanase Treatments on the Properties of Fresh and Frozen Doughs and Final Breads

Frozen bread doughs usually exhibit less bread volume and poor texture due to dough weakening as well as reduced yeast viability. The objectives of this study were to improve the textural properties of frozen bread dough by applying carbohydrate-active enzymes, α-amylase and endo-xylanase. Each enzyme was applied to dough formulation at 20 (748 and 3.5 units, respectively) and 100 ppm levels of flour, and their combined treatments were also applied. Enzyme-treated doughs were kept frozen at −20 °C for 2 weeks, and then baked following the official American Association of Cereal Chemists (AACC) method. A texture profile analysis of oven-baked breads was performed at 25 °C after a 5-day storage period. α-Amylase treatment at a 100 ppm level increased the specific bread volume by 24.5% and 21.9% when compared to untreated fresh and frozen bread doughs, respectively, and decreased crumb hardness by 63.4% and 58.3%; endo-xylanase (100 ppm) also decreased crumb hardness by 56.9% and 26.9%. The combined use of α-amylase and endo-xylanase retarded bread hardening synergistically after a 5-day storage period.

Introduction
The bakery industry has increasingly utilized the applications of freezing technology, and there are many papers on the storage of frozen doughs and the influence of such processes on the quality of the final product. The growing interest of the market toward frozen bakery goods is due to the economic advantage of a centralized manufacturing and distribution process as well as easier standardization of product quality. These products do not require specialized workers and raise the possibility of making "fresh" bread available at any time of the day [1]. Frozen dough has several baking problems such as poor bread volume and lower texture properties that have been ascribed to dough weakening and reduced yeast viability. The gradual loss of dough strength during frozen storage has been attributed to the reduction in gluten crosslinking caused by ice crystallization, the release of reducing substances from yeast, and the water redistribution provoked by a modification in the water binding capacity of dough constituents [2][3][4]. These phenomena may produce a loss in the gas retention capacity during fermentation, reflected by lower bread volume and an increase in fermentation time [4]. Thus, to obtain a product from frozen dough with a quality comparable to freshly made bread is a complex problem since the final structure is modified by several parameters [5]. To improve frozen dough, several technical modifications have been introduced in recent years. These include (1) the isolation of freeze-resistant yeasts [6]; the (2) addition of improvers such as emulsifiers and water-binding agents, e.g., hydrocolloids to stabilize the dough network; (3) the addition of wheat proteins to increase shelf life [7]; (4) the modification of dough composition [8]; (5) the use of heat stable enzymes to shorten fermentation time [9]; (6) the optimization of mixing, freezing and freeze-thaw cycles [10,11].
Enzymatic processes among various modification methods can effectively modify the structures of wheat polysaccharides, such as starch and non-starch polysaccharides, and the main components of cereals, which usually brings positive effects on dough and bread properties. The α-amylase (Aamyl) (EC 3.2.1.1) hydrolyses α-1,4-glycosidic linkages of starch and produces maltose, maltotriose, maltooligosaccharides, branched oligosaccharides, and low molecular weight α-limit dextrins. The added amylases increase the level of fermentable and reduce sugars in flour and dough, thus promoting yeast fermentation and the formation of Maillard reaction products, which in their turn, intensify bread flavor and crust color [12,13]. The molecular structure changes that occur in bread during storage which are responsible for the firming and staling phenomenon are quite complex and involve multiple constituents and mechanisms [14]. It is generally accepted that after the initial cooling process, the retrogradation/recrystallisation of starch, specifically of amylopectin branch chains in amylopectin, plays a major role in bread firming [12,14]. Starch retrogradation occurs in two different time-dependent stages [15,16]: amylose crystallization takes place after leaching out from starch granule in the short-term phase, while recrystallization through the outer branches of amylopectin is considerably slower than amylose retrogradation. Meanwhile, certain bacterial Aamyls have been known to decrease the rate of bread firming [17]. Endo-β-1,4-xylanases (EC 3.2.1.8.), also referred to as endo-xylanases (Exyl) or xylanases, have found widespread use in breadmaking applications since 1970s. Indeed, it has been well reported that the Exyls most suited for breadmaking are those active on the water-unextractable fraction and poorly active on the water-extractable fraction, solubilizing the insoluble arabinoxylan to give high molecular weight solubilized arabinoxylans and hence leading to a net loss of water holding capacity and an increased viscosity [18,19]. This action not only removes the insoluble arabinoxylan which interferes with the formation of the gluten network in dough, but also increases the stability of the dough system due to the increased viscosity. This in turn yields a more stable and flexible dough, which is easier to handle and gives improved oven spring, a larger loaf volume, improved crumb structure (fine crumbs and thin gas cell walls) and hence also a softer crumb [18]. Despite factual acceptance of Exyl as a means of improving dough and bread characteristics, their mode of action is still the subject of debate [20,21]. The objectives of this study were to understand the effects of Aamyl and Exyl treatment on the bread quality of frozen dough, which were compared with that of fresh dough.

Activity Determination of Endo-β-1,4-Xylanase and α-Amylase
Endo-β-1,4-xylanase and α-amylase activities were assayed by measuring the release of reducing sugar that were produced from beechwood xylan (Megazyme, Sydney, Australia) and soluble starch (Yakuri Pure Chemicals Co., Kyoto, Japan) hydrolysis, respectively. The endo-β-1,4-xylanase assay was carried out in 50 mM sodium acetate (pH 7.0) with 1% (w/v) beechwood xylan at 50 • C for 10 min. The α-amylase assay was performed in 50 mM sodium acetate (pH 5.0) with 1% (w/v) soluble starch at 50 • C for 10 min. The reactions were stopped by 500 µL of dinitrosalicylic acid (DNS) reagent, and then the reaction tubes were soaked in boiling water for 5 min. The absorbance of the solutions was measured at 575 nm. One unit of endo-β-1,4-xylanase and α-amylase was defined as the amount of enzyme required to release 1 µmol of reducing end per min. The activities of them were converted to the units of ppm because the enzymes were supplied as a form of powder. One part per million (ppm) of endo-β-1,4-xylanase and α-amylase was equivalent to 0.19 units and 37.4 units, respectively.

Bread-Making Procedure
Carbohydrate-active enzymes, such as endo-β-1,4-xylanase and α-amylase, were applied for bread-making. The bread dough formula consisted of wheat flour (300 g), dehydrated yeast (6.0 g), salt (4.5 g), sugar (18 g), shortening (9 g), ascorbic acid (200 ppm), and water (175.5 mL). The desired moisture content for dough mixing was determined based on the result of the Mixolab analysis (Chopin Technologies, Villeneuvela Garenne, France). The parameters obtained from the recorded curve were the amount of water required for the dough to produce a torque of 1.1 ± 0.07 Nm. The ingredients were mixed for 3.5 min at 25 • C in a 500 g pin mixer (National Manufacturing Co., Inc., Piscataway, NJ, USA). The dough was then divided into 150 g pieces. This fresh dough was fermented immediately while frozen dough was baked after a 2-week storage at −20 • C (without a given thawing time). These fresh and frozen doughs were fermented at 35 • C and 90% relative humidity (RH) for 180 min, and the dough samples were punched two times in the middle of fermentation at 105 and 155 min, respectively, and finally at 180 min once more just after fermentation. After the molding process of the doughs, they were proofed for 62 min and baked at 210 • C for 24 min in an oven (Daeyung Co., Seoul, Korea).

Extraction and Analysis of Water-Soluble Arabinoxylan in Bread Crumb
After the bread was baked in the oven and air-cooled on the lab table, the lyophilized and ground bread crumb sample was prepared, and it was suspended in deionized water (crumb:water = 1:5, w/v). Water-soluble solids were extracted from the resulting slurry at 5 • C for 1 h [5]. After centrifugation (5000× g, 4 • C and 10 min), the soluble extract was boiled and treated with thermostable α-amylase for 6 h. Once starch liquefaction was completed, the reaction mixture was cooled to 60 • C, and the protease treatment was pursued at 60 • C for another 6 h to remove proteins. After the enzyme-treated samples were boiled for 20 min, its pH was adjusted to 4.3 by adding 200 mM of sodium acetate buffer containing 1% (w/v) sodium azide. By adding amyloglucosidase to the sample solution, starch fragments were completely hydrolyzed at 60 • C for 24 h, which was centrifuged at 10,000× g for 10 min. To the supernatant, five volumes of ethanol were added and held at 4 • C overnight. The precipitate was washed twice with 80% ethanol and was allowed to dry in the air until no solvent could be detected by odor. This purified water-soluble arabinoxylan (WS-AX) was dissolved in water at 60 • C for 2 h, filtered through a polypropylene membrane filter (0.45 µm pore size), and then injected (100 µL) with 10 mg/mL to the HPLC system for the analysis of structural changes in the WS-AX component.

Analysis of Bread Loaf Volume and Crust Color
The loaf volume of the bread sample was determined with a Volscan Profiler (Stable Microsystems, Godalming, Surrey, UK). The crust color of the bread was determined according to the AACC Approved Method 14-22 (AACC International, 2000) using the Minolta chromameter (CR-300, Minolta Camera Co. Ltd., Osaka, Japan). The chromameter was calibrated with a Minolta calibration plate, and the C standard illuminant was used. Data were expressed as lightness/darkness (L*), redness/greenness (a*), and yellowness/blueness (b*). The crust color was measured on the top of each bread sample in triplicate.

Texture Profile Analysis of Bread Crumb
Textural characteristics of bread sample were examined just after cooling the bread and at 5 days after baking by the texture analyzer (TMS-Pro, Mecmesin Ltd., West Sussex, UK) equipped with a 25-N load cell. The average values determined from three different loaves were reported as data points. At least two slices of bread (20 mm thickness each) cut from each loaf center were applied for this analysis. The bread slices were compressed up to 14 mm (equivalent to 70% of slice height) from the cross section of bread crumb with a 25 mm diameter cylindrical probe at a test speed of 60 mm/min. The crumb firmness and elasticity were evaluated by applying compression two times in each cycle.

Statistical Analysis
ANOVA and Tukey's honestly significant difference tests were performed using Sigmaplot software package 13.0 (Systat Software Inc., San Jose, CA, USA).

Molecular Weight Distribution of Water-Soluble Arabinoxylan (WS-AX) Extracted from the Bread Crumb
To improve the quality of frozen bread dough (FZDB; dough that was frozen at −20 • C for 2 weeks), the enzymes, α-amylase (Aamyl) and endo-xylanase (Exyl), were formulated in the dough making. Water-soluble arabinoxylans (WS-AXs) were extracted from the enzyme-treated bread crumb, and their relative Mw distributions were analyzed by HPSEC. As the result of increasing the dose level of Exyl activity in both the fresh dough bread (FSDB) and the FZDB, the Mw of WS-AX noticeably decreased (Figure 1a,b). The increase in the dose level of Aamyl activity in both FSDB and FZDB did not significantly change the AX structure from the control as expected from the substrate specificity of Aamyl ( Figure 1). Even in the dough samples treated with both Aamyl and Exyl, it was clearly shown that Aamyl did not affect the size of WS-AX (Figure 1c,d). Even though the action of Exyl might not be significant during the freezing storage period, the enzyme remained stable and could be well activated by thawing, and thus the reactivated Exyl probably hydrolyzed AX during mixing, fermentation, and baking of the FZDB (Figure 2). In comparison to FSDB, the FZDB clearly displayed less hydrolysis of WS-AX but still significant decrease in Mw of WS-AX was observed. It could be expected that reactivation of the enzymes in frozen dough might take an induction period and low temperature storage could inactivate a certain part of the enzyme, which delayed the hydrolysis of the AX structure. It was previously reported that the Mw reduction in WS-AX led to the decrease in dough viscosity [22] and AX-based oligosaccharide products had a health-beneficial effect as a prebiotic [23].

Effects of Carbohydrate-Active Enzyme Treatment on Wheat Bread Quality
To determine the effects of Aamyl and Exyl, which are active enzymes in starch and cell wall polysaccharides, respectively, the quality of breads made from the FSD and FZD was evaluated by analyzing the quality parameters such as loaf volume, textural properties, and crumb color of the breads. The loaf volume of bread affects consumer preference on product purchase as one of the important physical properties of bread. The specific volume of untreated FZDB (4.84 mL/g) was smaller by 8.4% than that of FSDB (5.27 mL/g) (Figure 3). Not only between the untreated controls, but the comparison between enzyme-treated FSDB and FZDB also showed that the specific volume of FZDB was consistently lower than that of FSDB at the same enzyme treating levels. This smaller specific volume of FZDB might be due to its lower gas retention capability through a weaker gluten network by ice crystals formed in the freezing process. Moreover, the viability of yeast cells in thawed dough was negatively affected by freezing storage, and less CO2 production during baking resulted in incomplete pore swelling and a reduction in the specific volume of bread when compared to fresh baked bread [24,25].

Texture Profile Analysis of Enzyme-Treated Bread Crumbs
Textural analysis of food materials is an important technical tool for evaluating consumer acceptance on the processed products. The parameters obtained from food texture analysis seems to be indirectly representing most of the critical attributes affecting the organoleptic properties. In case of oven-baked goods, its crust and crumb are generally recognized as the objects for the texture analysis. Fresh oven-baked bread displays a firm crust while its crumb exists as a porous structure induced by CO2 gas released by yeast fermentation, and these final air cells in the crumb are mainly interconnected by a gluten-starch network [28].
When the breads were treated with carbo-active enzymes, the hardness and chewiness of FSDB decreased to 1.24 N and 0.96 J with a high dose of Aamyl (A100), respectively, and decreased to 1.46 N and 1.17 J with a high dose of Exyl (E100) as well, from 3.39 N and 2.60 J of these parameters of control bread. When combined treatment of a high dose of both Aamyl and Exyl (A100E100) was applied to fresh bread dough formulation, these parameters decreased to 1.19 N and 0.93 J, respectively, of which the result showed somewhat decreasing effects on both hardness and chewiness but displayed no statistically significant differences (Table 1). Furthermore, Aamyl (A100) seemed to act more effectively than Exyl (E100) did in relation to the textural change in the breads. The hardness of the crumb is closely related to the structure of gas cell walls, especially, which is affected by the size and number of gas cells. It was reported that the larger size of gas cells by adding Aamyl resulted in bread with a softer crumb [26,29]. It was also previously reported that a small change in the loaf volume of bread might cause a large difference in crumb hardness [30]. Indeed, the crumb hardness of untreated control FSDB was almost three times greater than that of FSDB with high doses of Aamyl and Exyl (A100E100) whereas the volume difference between them was only 25% in our present study. Aamyl was known to partially hydrolyze amylopectin chains in starch molecules, and thus decreased the degree of its recrystallization and prevented water molecule fixation inside retrograded starch structures [31][32][33]. In this study, we also investigated the effect of enzyme treatment on the specific volume of FSDB and FZDB. As a result, both in FSDB and FZDB, Exyl treatment did not significantly change it from the control as expected from the previous study [26]. When 20 and 100 ppm (flour weight basis) of Aamyl were applied in the dough formulation, the increase in specific volume was observed by 12.7 and 24.5%, respectively, in FSDB ( Figure 3). Meanwhile, 15.3 and 21.9% of volume increases were obtained, respectively, by adding 20 and 100 ppm of Aamyl from the FZDB. When Aamyl and Exyl were treated together, a volume increase by Aamyl was still clearly shown, but Exyl effect on the volume was not detectable at all. Accumulation of fermentable reducing sugars by Aamyl action accelerated CO 2 production by yeast, which resulted in larger air cells by high gas pressure in the dough. In the case of Exyl-treated dough, it was generally reported that this enzyme treatment also improved the bread volume but no significant change in the volume was observed in our study. This inconsistency might be due to the variation of the amount and properties of pentosans depending on wheat cultivars and thus a different action pattern of Exyl on a wide range of these substrates [27]. In addition, it was reported that a decrease in pentosan content or destruction of pentosan did not affect bread volume as well [26].

Texture Profile Analysis of Enzyme-Treated Bread Crumbs
Textural analysis of food materials is an important technical tool for evaluating consumer acceptance on the processed products. The parameters obtained from food texture analysis seems to be indirectly representing most of the critical attributes affecting the organoleptic properties. In case of oven-baked goods, its crust and crumb are generally recognized as the objects for the texture analysis. Fresh oven-baked bread displays a firm crust while its crumb exists as a porous structure induced by CO 2 gas released by yeast fermentation, and these final air cells in the crumb are mainly interconnected by a gluten-starch network [28].
When the breads were treated with carbo-active enzymes, the hardness and chewiness of FSDB decreased to 1.24 N and 0.96 J with a high dose of Aamyl (A100), respectively, and decreased to 1.46 N and 1.17 J with a high dose of Exyl (E100) as well, from 3.39 N and 2.60 J of these parameters of control bread. When combined treatment of a high dose of both Aamyl and Exyl (A100E100) was applied to fresh bread dough formulation, these parameters decreased to 1.19 N and 0.93 J, respectively, of which the result showed somewhat decreasing effects on both hardness and chewiness but displayed no statistically significant differences (Table 1). Furthermore, Aamyl (A100) seemed to act more effectively than Exyl (E100) did in relation to the textural change in the breads. The hardness of the crumb is closely related to the structure of gas cell walls, especially, which is affected by the size and number of gas cells. It was reported that the larger size of gas cells by adding Aamyl resulted in bread with a softer crumb [26,29]. It was also previously reported that a small change in the loaf volume of bread might cause a large difference in crumb hardness [30]. Indeed, the crumb hardness of untreated control FSDB was almost three times greater than that of FSDB with high doses of Aamyl and Exyl (A100E100) whereas the volume difference between them was only 25% in our present study. Aamyl was known to partially hydrolyze amylopectin chains in starch molecules, and thus decreased the degree of its recrystallization and prevented water molecule fixation inside retrograded starch structures [31][32][33]. Table 1. Texture profile analysis results of enzyme-treated fresh and frozen bread doughs at storage of day 0 † . In this experiment, the texture of baked FSDB and FZDB was analyzed after 5 days of storage at room temperature. From the control breads, the hardness of FSDB and FZDB were measured to be 7.22 and 9.59 N, respectively, which showed that the hardness of FZDB increased more rapidly than that of FSDB along with storage time ( Table 2). Water molecule migration, and ice crystal formation and recrystallization during the freezing-storage period of FZDB might cause the starch component to restructure, in turn leading to the bread hardening process due to the changes in gelatinization and retrogradation patterns of starch molecules [34]. Along with the retrograding process, water molecules could move into an amylopectin crystal structure and resided inside. These crystalline hydrate water molecules might not have the capability of network formation, which resulted in disrupted flexibility of the gluten network and increased crumb hardness [14]. As a result, the degree of retrogradation substantially increased as the storage time was extended in the freezing condition [3,35,36]. Water-unextractable arabinoxylan (WU-AX) is one of the important polymeric materials maintaining the structural integrity of cereal endosperm cells. When this cell wall component is disrupted by one of hemicellulose-type enzymes, xylanase, the crumb hardness is subjected to decrease by its plasticizing effect on the gluten-starch network in the breads [27,37]. The antistaling effect of xylanase on breads was observed by DSC analysis previously, which suggested that xyalanase action partially prevents the retrogradation of the starch component in the beard crumb [14,38,39].

Effects of Enzyme Treatment on the Color of Bread Crust
The color of bread crust is one of the important quality factors affecting consumer preference expected from bread products. In general, bread crust is characterized as low moisture and dark brown in color. The typical bread color on the crust is generated by chemical reactions such as the Maillard reaction and caramelization. As the dosage level of Aamyl increased in the bread dough formulation, reducing sugar formation was accelerated and the released sugars were utilized for the Maillard reaction. The resulting outcome displayed low L* values and led to a much darker crust formation. The crust color between light and dark brown was preferred by most consumers, and the flavor and taste of baked bread could be improved as the result of the Maillard reaction. Exyl also produced reducing sugars by hydrolyzing AX and decreased the L* value through the Maillard reaction. However, approximately 2% of the AX content was relatively low in wheat flour and a limited amount of reducing sugar could be released by this enzyme reaction, and thus much less browning color was developed compared to an Aamyl reaction on starch that consists of 70-80% of wheat flour [40,41]. Between the untreated FSDB and FZDB controls, overall decreases in all the L*, a*, and b* values were significantly larger from FZDB when compared to FSDB; L* value decreased from 50.18 of FSDB to 42.80 of FZDB (Table 3). In the process of freezing and thawing of wheat dough, water exudation by syneresis concentrated the amount of wheat ingredients in dough, and the browning reaction might be facilitated, which in turn resulted in the darker color of bread crust. In addition, fermentation might not be complete due to the loss of yeast activity and unfermented sugars that remained, and thus the residual sugars possibly made a darker color due to the browning reaction during the baking process of frozen dough [42].  In this study, it was clearly shown that α-amylase treatment of bread dough resulted in the improvement of bread quality significantly. In addition, the endo-xyalanase action also partially assisted the prevention of quality loss in the beard crumb. Thus, positive effects of these enzymes on frozen bread dough can be promisingly utilized to prevent the quality loss of cold-chain distributed bread products. In terms of wheat cultivar development, the endogenous enzyme activities of wheat grains could be enhanced and thus the extra incorporation of exogenous enzymes might not be required to maintain bread quality.


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Density of Densified Paraserianthes falcataria Wood Pre-treated with Alkali

Wood densification is done to improve its mechanical and physical properties, by compressing the porous structure under steam, heat and pressure called Viscoelastic-Thermal Compression (VTC). This study had been done to evaluate the effect of alkaline pre-treatment using different concentrations of Sodium hydroxide (NaOH) on Paraserianthes falcataria (local name: Batai) and its density. Alkaline pre-treatment was done before the densification process, using three different concentrations of NaOH (3%, 6% and 9%) with 0% NaOH as the control. There were 30 replicates used for the study. The lamina was prepared by trimming the lamina sawn timber into specific size. Then, laminas cooking process in different NaOH concentrations for 30 minutes, followed by hot water extraction with acetic acid for 20 minutes and 24 hours of oven dried under temperature 103 ± 5°C. The laminas were then pressed using hot press machine under 105°C temperature and 6 MPa pressure for 30 minutes before cooling phase were conducted for 10 minutes. The density was evaluated continuously for 7 days and the results showed that densified P. falcataria without alkaline pre-treatment (0% NaOH) has the highest density compared to treated densified P. falcataria with different alkaline pre-treatments (3%, 6% and 9%); meanwhile the highest concentration of NaOH (9%) has the lowest density value compared to 3% and 6%, as 3% has the higher value than 6% NaOH.

Introduction
The concept of wood densification was first invented in 1900s. There are many studies had been done on wood densification using different wood species and produce many results. Wood densification is a process where wood compression, impregnation of cell lumens with liquid or both was done as to increase the wood density. Viscoelastic-Thermal Compression (VTC) of wood is a process to increase the density of the material properties [1]. Wood compression against the wood grain under extreme heat and pressure condition are included in VTC process. Densification can be achieved when the wood is softened by heat and compressed with pressure.
It is hard to compress a natural wood into compact and compress wood without eliminating lignin. The void in cell wall after the lignin removal after alkali pre-treatment lead to porous cell wall and can be easily compacted during pressing [2]. There were several wood compressions had been reported over years ago. Previous study shows that high value of wood produced from wood densification process is suitable for construction material, strong as metal and alloy materials. Densification allowed low-density wood to be substitute for harder species as the species can be modified from low-density into high performance and high value product [3]. Wood species that already with high density can be  [4]. However, this study is to shows the effects of lignin removal on wood densification.

Materials and Methods
For this study, Paraserianthes falcataria (Local name: Batai) were obtained from Sapulut Forest Development Sdn. Bhd. P. falcataria is a fast-growing large tree and can grow up to 40m tall. This species widely planted in tropics country such as Indonesia and Malaysia.
For sample preparation, wood laminas of P. falcataria were collected random (sapwood and heartwood) as to avoid biasness in results. The woods were trimmed to 300 mm (length) x 50 mm (width) x 20 mm (thickness).
For the alkaline pre-treatment, Soda Pulping method for Pulp & Paper Technology (2016), was used, where Sodium hydroxide, NaOH was used at different concentrations (3%, 6%, & 9%) with 0% NaOH as control. Wood laminas had been treated in closed chamber in the fume hood for safety precautions. Then hot water extraction was done for 20 minutes, after a fixed amount of acetic acid (GAA) were added to neutralize the NaOH, to wash out the remaining NaOH (until the water turned clear). The laminas were then oven-dried under temperature 103 ± 3 °C for 24 hours and undergone conditioning phase. After that, the treated laminas were compressed using hot press at 6 MPa and 100°C for 30 minutes with 8 mm stopper as targeted final thickness. Cooling phase had been done for 10 minutes before the compressed wood being taken out from the machine. There are 30 replicates for each parameter. First reading had been measured 15 minutes after the cooling phase. The densities of the compressed woods were studied for 7 days continuously and plotted.
In this study, lengths, widths, and thicknesses of all the woods were measured using Vernier caliper and the mean values were recorded as to calculate the volume (V) of the laminas. On other hand, the weight (W) of the laminas were also measured using weigh balance. Then, the density of the laminas was calculated according to Equation 1: Where, W = weight of lamina (kg) V = volume of lamina (m 3 )

Results and Discussion
Generally, density is simple to decide and very much associated to numerous other physical and mechanical properties of wood, such as strength and stiffness. Density is also utilized as a wood quality value indicator, where related to various purposes. For example, wood with high density and strength is used for construction; meanwhile low-density wood is suitable for pulp and paper [5]. Wood density can be affected by many factors, such as wood species, chemical composition in the wood naturally, age of the tree, part of tree used and the wood processing pattern.
The results for this study had been showed as a graph where, Figure 1 shows that the higher concentration of NaOH usage in alkaline pre-treatments lead to lower wood density, where densified wood with 9% NaOH has the lower density compared to untreated densified wood with 0% NaOH.
According to previous study, Alkaline pre-treatment leads to removal of lignin and hemicellulose content in natural wood, whereas the wood will become more porous and less rigid [2]. The springback occurs in the wood after compression might influenced by the absence of the lignin [6] as lignin act as the binder that bind the cellulose and hemicellulose together [7]. Alkaline treatment degrades certain amount of hemicellulose, lignin, wax, and oils of fiber cell wall. Reduction of lignin could weaken the particle strength as the lignin acts as binder of cellulose fibers [8].
Wood compression resulting in reduced of thickness to about 40% as the wood lumen and porous wood cell had collapsed [2]. High density wood will be used for structural application where resistance is important. From the graph in Figure 1, the density of untreated wood before densification shows very low in density, as not more than 400 kg/cm 3 , however after the densification process, the density of the untreated wood shows drastic increases in density from344.6 kg/cm 3 to 909.2 kg/cm 3 on day 7 th . The density of treated wood after compression also had showed major changes in density, as the highest concentration, 9% NaOH has the lowest density compared to 3% and 6% NaOH. From my study, lignin played the major role in wood modification especially wood densification. Alkaline pretreatment diminished hemicellulose, lignin, wax and oils of fibre cell wall. Removal of lignin might weaken the properties as the lignin used as the binder [8]. of NaOH. Figure 2 shows the densified P. falcataria with and without alkaline pre-treatment after 7 days observation. From the Figure 2, the densified woods show slightly differences in thickness as all the woods were compressed using 8 mm stopper. Elastic deformation recovered after load was removed from the densified wood. Continuous deformation can be recovered when the densified woods were exposed to humidity when there's so external force applied [6]. This is called spring-back. Wood is well-known as viscoelastic material and the mechanical properties depend on time, heat and environment humidity. Alkaline pre-treatment was done to eliminate lignin from the cell wall of the wood. Chemical pre-treatment, such as alkaline pre-treatment, leads to lignin and hemicelluloses content elimination from cell wall. The wood becomes more porous and less rigid when lignin and hemicelluloses partially removed from the cell wall [2]. When the hot-pressing to opposite to the wood growth direction, the cell wall without lignin breakdown altogether. This process caused the densified wood to reduce in thickness for about 40% (Figure 3) and increased in density.  NaOH, (c) 3% NaOH, (d) 0% NaOH (control). It is hard to compress a natural wood without lignification into a compact densified wood [2]. However, the same studies showed that after the removal of lignin from the natural wood, the wood become brittle and easily crushed during pressing, probably because of the delignification process, as lignin is used as a binder in the cell wall [7]. Wood density is one of the wood qualities which corelated to other properties, such as toughness, stiffness and performance in use [5].

Conclusion
The densification procedure, where plasticizing of the wood, is considered as VTC densification process. In this preliminary study, the density of the densified P. falcataria with and without alkaline pre-treatment had been studied for 7 days, where high amount of NaOH used to delignify the wood laminas lead to lower in wood density.


s2fieldsofstudy:
Agricultural And Food Sciences
Environmental Science

text:
Sulfur Amendment of Soil Improves Establishment and Growth of Firs in a Field Naturally Infested with Phytophthora

Acidification of soil from pH 6 to 4 by incorporating elemental sulfur reduced mortality and improved color and initial growth of Fraser fir, Abies fraseri (Pursh) Poir., and Canaan fir, Abies balsamea (L.) Mill. var. phanerolepis Fernald, planted into a field that had previously experienced significant losses consistent with phytophthora root rot. Acidifying the soil improved tree color starting the year of planting and persisting through five years. During their second year after planting, extension of terminal growth was 12.5 vs. 5.6 cm (4.9 vs. 2.2 in) for plots with soil pH of 4 and 6, respectively, averaged across tree species. In subsequent years, the growth rate of trees was unaffected by having acidified the soil. Over the course of five years, the average annual mortality rate for the trees was 1.4, 4.0, 9.7, and 12.2% for Canaan fir (pH 4), Canaan fir (pH 6), Fraser fir (pH 4), and Fraser fir (pH 6), respectively. However, all tree mortality for Canaan fir planted into acidified soil occurred during the first two years. A root dip with potassium phosphite at the time of planting only benefitted Fraser fir. Minimizing losses of trees in this field would require planting species less susceptible or resistant to phytophthora root rot infection and soil acidification. Index words: soil acidification, phytophthora root rot, potassium phosphite, root dip. Species used in this study: Fraser fir, Abies fraseri (Pursh) Poir.; Canaan fir, Abies balsamea (L.) Mill. var. phanerolepis Fernald. Chemicals used in this study: acibenzolar-S-methyl (Actigard 50WDG); clothianidin (Arena 50WDG), imidacloprid (Xytect 2F), potassium phosphite (Helena Prophyt), sulfur. Significance to the Horticulture Industry Losses due to root rots caused by Phytophthora species can be difficult to avoid where the pathogen is present and soils are conducive to disease. For Christmas tree plantations, planting less susceptible Canaan fir in place of the highly susceptible Fraser fir reduced losses by twothirds. Reducing soil pH from 6 to 4 reduced losses among Canaan fir by an additional two-thirds. The combined actions reduced tree mortality by 89%.

Introduction
True firs (Abies: Pinaceae) are especially valued as Christmas trees due to superior needle retention, attractive foliage, and pleasant aroma. However, firs are susceptible to many soil-dwelling pests, including root rot diseases caused by various Phytophthora species Chastagner 2012, Li et al. 2019), which are water molds belonging to Oomycota. Under adverse conditions, these organisms form thick-walled oospores (the result of sexual reproduction, usually produced by recombination among differing mating types) and chlamydospores (produced asexually). These spores may remain viable in soil for several years. When conditions are wet, both spore types germinate to develop sporangiophores with sporangia. These, in turn, break open to release zoospores, a free-living life stage with two flagella that can move within a film of water in soil to roots of potential hosts. Upon contact with the root of a suitable host, the zoospore detaches its flagella and forms a cell wall in a process called encystment, rapidly followed by germination of the cyst, from which threadlike hyphae invade the roots. The life cycle is complete when Phytophthora hyphae form either sporangia, oospores, or chlamydospores (Parke and Eberhart 2016). Christmas tree farms in New England are faced with two factors that exacerbate tree losses due to root rot. Soil conditions (soil texture, depth, slope, and drainage) are so variable that even small fields of less than one hectare often contain more than one soil type. This leads to difficulty with losses from root rots when susceptible species are planted in areas (typically, finer textured soils or areas that are poorly drained) more conducive to disease development. Secondly, this region is predicted to receive greater and more variable rainfall resulting from anthropogenic climate change (IPCC 2007), in which precipitation may occur in larger events (e.g., from tropical storms or from persistent low-pressure systems). Conditions leading to more frequent soil saturation events suggests that losses of Christmas trees from Phytophthora may increase over time in this region unless we can find effective management options.
Phytophthora root rots of Christmas trees in Connecticut include several species, including P. abietivora, P. cactorum, P. kelmania, P. pini, and P. plurivora (McKeever andChastagner 2016, Li et al. 2019). World-wide there are many more species of Phytophthora found to infect Christmas trees (Talgø et al. 2007, Li et al. 2019. Differences among these species with respect to their ability to infect the roots of various species of fir trees are not known, or of the variations among these species that define conditions especially conducive or restrictive for their growth. Current management techniques involve buying healthy plants, using raised beds or installation of drainage tiles, avoiding planting highly susceptible fir species (e.g., Fraser fir) in areas of wet soil or where disease has previously occurred, and either growing resistant species (A. bornmuelleriana Mattf. [Turkish fir] or A. firma Siebold & Zucc. [Momi fir]), or grafting susceptible trees as scions on these Phytophthora-resistant rootstocks (Frampton and Benson, 2012). Use of fungicides has limited practical value under conditions conducive to disease (Benson et al. 2006, Thomas andPickel 2011).
Methods of growing trees that prevent phytophthora root rots should also enhance the establishment and growth of fir trees, because healthy root systems allow properly functioning plants to reach their full genetic potential. A complete assessment of methods to prevent root rot occurrence should start with the disease triangle: plant disease occurs only when there is co-occurrence of disease inoculum, a susceptible host, and environmental conditions conducive to infection (Francl 2001). Any one of the three sides to the disease triangle could be manipulated to decrease disease.
One method to manipulate the soil environment is to change the soil pH. Previous research has demonstrated that reducing soil pH to about 4 can protect various species from infection by phytophthora root rots, attributed to the negative effects of hydrogen ions on development and germination of sporangia, and survival of oospores and chlamydospores (Kong et al. 2009). Fir species native to eastern North America are adapted to highly acid soils (Richter et al. 2011) and so acidification of soil to a pH of 4 can be anticipated to favor growth and survival of Abies over Phytophthora.
Reducing host susceptibility might be accomplished by growing resistant or less susceptible species or varieties, or by inducing resistance in otherwise susceptible hosts. Induction of the systemic acquired resistance pathway can be accomplished with phosphite-containing products, the insecticides imidacloprid or clothianidin, which are metabolized to form salicylate agonists (Ford et al. 2010), or acibenzolar-S-methyl (Cole 1999). In an earlier study, root dips with products that are known to be able to induce the systemic acquired resistance pathway in plants improved the health of Christmas trees transplanted into a field infested with phytophthora root rot pathogens: Douglas-fir and Fraser fir appeared to benefit from root dips containing potassium phosphite, while Canaan fir and white spruce did not (Cowles et al. 2012).
This work focuses on interactions between host susceptibility, by (1) comparing two species, and (2) using products in a root dip at the time of planting that should activate the systemic acquired resistance pathway, and manipulation of the soil pH. I explored how well we would be able to establish and grow Christmas trees on a site suspected to be unusually uniformly infested with Phytophthora.

Materials and Methods
A field trial planted in 2010 investigated the growth performance of exotic firs at a cooperating farm site in Brooklyn, CT, where the grower had experienced losses consistent with the presence of Phytophthora root rot (C. Langevin, personal comm.). By October of 2013, all 60 Fraser firs planted at this site had died, whereas there were few losses among the 660 Turkish, Nordmann, or Trojan firs (Abies bornmuelleriana, A. nordmanniana (Steven) Spach, and A. nordmanniana (Steven) Spach ssp. equitrojani Mattf, respectively) (R. Cowles, unpublished data). During May of 2014, the trees from this experiment were removed, including main roots uprooted with the trunks, so that the field could be repurposed for additional experiments. Soils in this field are Paxton and Woodbridge fine sandy loams, averaging 5.5% slope. These are typically well-drained or moderately well-drained soils with a pH of 5.2 to 5.5 within the surface 15 cm (6 in), and are classified as prime farmland (https://casoilresource.lawr.ucdavis.edu/ gmap/). The short-term history of this field had been Christmas tree production, prior to which it had been used for growing field corn. A history of lime application to grow corn probably explains the soil pH of 6.0 to 6.2 measured in 2014.
To prepare this field for experiments involving soil pH manipulation, pelletized sulfur (Winfield Solutions, St. Paul, MN, containing 90% elemental sulfur) was spread at a rate of 3,370 kg . /ha À1 (3,007 lb . /A À1 ) of product on June 18, 2014 into randomized main plots, with each plot sufficiently large (3.4 by 11.7 m) (11.1 by 38.4 ft) to plant 14 trees at a spacing of 1.7 m (5.6 ft) within the row and 1.7 m between rows. There were 23 replicates of paired main plots, either receiving no sulfur or 16.8 kg (37 lb) of sulfur product per plot. Sulfur was manually spread within plots, and then incorporated with a rototiller to a depth of 15 cm (6 in) on June 18, 2014.
The overall experiment was a 2 3 2 3 7 factorial splitsplit plot randomized complete block design with 23 replicates to investigate the influence of soil pH (consisting of two levels which were main plots of a split-plot design, sulfur or no sulfur), species (two disease susceptibility levels, representing split-split plots), and various root dips (seven levels randomized within subplots) on the initial establishment success and growth of bare-root Christmas trees, for a total of 28 treatment combinations. The seven root dip treatments included a 3 3 2 factorial evaluation of insecticide times potassium phosphite interactions, plus a treatment not balanced with the factorial design to investigate effectiveness of a root dip with acibenzolar-Smethyl (See Table 1). There were 644 trees individually treated at the time of planting: 322 each of Canaan fir (less susceptible to root rot) and Fraser fir (highly susceptible to root rot). Three-year old 30-cm (11.8 in) tall bare root transplants were obtained from Reliable Source Nursery (Star City, WV) and planted April 13 and 14, 2015. Root dips involved submerging the root system into 8 L (2.1 gal) of prepared solutions held in a 20 L (5.3 gal) bucket, and allowing excess liquid to drain back into the bucket immediately prior to planting. Trees were planted into holes, which were backfilled with soil while positioning the root crown just below the soil surface, and then the soil compressed by stepping on the soil to guarantee good soilto-root contact.
Composite soil samples were taken to be representative of the rooting zone from each plot on August 31, 2015 and again in September, 2016. Soil pH was measured in the soil testing laboratory at the Valley Laboratory of the Connecticut Agricultural Experiment Station. Deionized water was added to soil to form a slurry, which was allowed to stabilize for about 20 min before taking measurements with a previously calibrated Accumet XL25 pH meter (Fisher Scientific, Hampton, NH). Soil pH measurements from main plots were used to confirm the effectiveness of the sulfur application and the stability of these effects.
Tree condition was evaluated on September 15, 2015, August 17, 2016, September 26, 2017, July 19, 2018, and April 15, 2019. Trees were assigned health ratings according to the following scale: 0, dead; 1, almost dead (yellowed and wilting); 2, very yellow; 3, slight yellowing; 4, fully green. A similar (but mirrored numbering) disease rating system was used in NC to assess Fraser fir root health and underlying phytophthora root rot (Richter et al. 2011). The idea behind the ratings is that they are providing a measure of underlying root health, without taking destructive samples of roots. The length of the current year's growth of the central leader was measured once each year to the nearest cm.
Ten dying Fraser fir trees were sampled in October 10, 2017 to confirm whether the plant losses were due to Phytophthora root rot infections. Diseased plants were taken to the laboratory to isolate the causal agent for identification purposes.
Soil pH data were analyzed via t-tests in Excel 2013 (Microsoft, Redmond, WA). Tree health ratings, mortality, and growth were assessed through analysis of variance (ANOVA) with Statistix 9 (Analytical Software, Tallahassee, FL). Analyses of tree health ratings were conducted twice: once with all subplot treatments (including the acibenzolar-S-methyl treatment), and once by excluding the acibenzolar-S-treatment to investigate the remaining factorial design. The general ANOVA feature was used to construct models that followed a split-split plot design, with or without the underlying subplot factorial treatment combinations in subplots.

Results and Discussion
Previous losses in this field were assumed to have been due to phytophthora root rot. Dying trees taken to the laboratory in October, 2017 were used to isolate the disease-causing organism. Phytophthora was recovered from stem lesions typical of infection by this genus. The unusually thick oospore walls indicated that it was an undescribed species, and so Koch's postulates were tested to prove its pathogenicity and molecular genetics used to assist in its phylogenetic placement. The full description of the species, Phytophthora abietivora D.W. Li, N.P. Schultes, J. A. LaMondia, R. S. Cowles, were then published (Li et al., 2019).
The soil pH measurements taken in 2015 and 2016 demonstrated dramatic acidification within one year following sulfur incorporation. The incorporation of sulfur had the intended effect of reducing pH, as the pH values for the sulfur-incorporated plots vs. no-sulfur plots were 4.06 6 0.09 (mean 6 se) and 5.95 6 0.06, respectively, as measured in August, 2015. Measurements of soil in 2016 were on average 0.4 pH units higher than in 2015, with values of 4.5 6 0.09 and 6.2 6 0.09; the cause for the shift in pH measurements from the previous year is not known. The pH was not reduced as much in the center of the field where the Paxton soil type occurred (average reduction of 1.6 pH units, n ¼ 13), as in the Woodbridge soil type (reduction of 2.0 pH units, n ¼ 10; P , 0.05, t-test of two populations, averaged over measurements from both years).
In the year of planting (2015), the application of sulfur significantly improved the color of transplants (F (1,22) ¼ 18.9; P , 0.001). Canaan fir had better color than Fraser fir (F (1,45) ¼ 9.08; P , 0.01); sulfur incorporation improved the health of both species to a similar degree (sulfur 3 species interaction F (1,44) ¼ 2.61, P ¼ 0.11). The insecticides imidacloprid and clothianidin applied in root dips did not enhance plant health (F (2,447) ¼ 0.39, P ¼ 0.67). The only root dip factor providing statistically significant benefit was the use of potassium phosphite, which only benefitted the Fraser firs (phosphite main effect, F (1,447) ¼ 3.51, P ¼ 0.061; species 3 phosphite interaction, F (1,447) ¼ 5.03, P , 0.05) (Fig. 1). This phosphite 3 species interaction for Canaan and Fraser firs is consistent with the response reported earlier (Cowles et al. 2012). Potassium phosphite is known to reduce Phytophthora infection by activating the systemic acquired resistance (SAR) biochemical pathways in plants (Machinandiarena et al. 2012). The different responses of Fraser and Canaan firs to a phosphite root dip could be explained if the SAR pathway is inducible in Fraser fir, but constitutively active in Canaan fir. The influence of soil acidification on the probability of tree death was marginally statistically significant for Canaan fir. Plots with pH 4 vs. 6 soil experienced 3 vs. 11 trees dying out of a starting population of 161 trees per group (P ¼ 0.052, Fisher's Exact test). For Fraser firs, this comparison was 21 vs. 27 trees dying (P ¼ 0.43).
In the subsequent year (2016), soil acidification resulted in significantly less cumulative mortality in both Canaan fir, with 6.8 vs. 15% in pH 6 soil, and Fraser fir, with 20 vs. For surviving trees, the color of the trees was greatly improved over 2015 (the mean color rating was between 3.5 and 4.0 for all groups in 2016. The growth of leader terminals in the sulfur-amended plots averaged about 12.5 6 0.5 cm for both species, and about half that much (5.6 6 0.3 cm) in no-sulfur plots, which were highly significantly different (F (1,22) ¼ 37.5, P , 0.001) (Fig. 2). The effects of root dips at the time of planting manifested themselves as contributing to differences in terminal growth in 2016. Of these root dip treatments, only the acibenzolar-S-methyl treatment was significantly different from the water check, and it significantly decreased the terminal growth (F (3,428) ¼ 5.8, P , 0.001) (Fig. 2). Further experiments would be necessary to determine whether the dose of acibenzolar-S- methyl was phytotoxic, and whether lower doses in a root dip could be beneficial to root health. The patterns for tree color, growth, and mortality are presented in Fig. 3 for the course of this experiment. For the third through fifth years after planting, the ranking of color qualities for the four treatment combination groups (Canaan vs. Fraser, and acidified vs. non-acidified soils) remained stable, with the exception of the last year of measurements. In 2019, Fraser firs with acidified soil improved in color, whereas color ratings for Canaan fir growing in higher pH soil decreased. This field had not received nitrogen fertilizer, and a decline in the color of the Canaan firs from 2017 to 2019 may reflect nitrogen deficiencies. The improvement in the color of the Fraser firs from 2018 to 2019 may reflect, to some degree, the loss of trees that had previously received poor color ratings, that subsequently died, and were no longer used in calculating the color rating (a color rating of zero for a dead tree was only used once, and the individual was removed from further ratings). The growth and mortality rates for these four treatment combination groups remained nearly constant from 2016 through 2019, as indicated by the nearly linear results through this period (Fig. 3). The mortality rates from 2016 to 2019 did not significantly differ between acidified and non-acidified soil for Fraser fir (P ¼ 0.54, Fisher's Exact test), and so the bulk of the benefit to this species occurred within the first two years of planting, when the mortality probabilities did differ (P ¼ 0.0056, Fisher's Exact test). It is notable that there was no additional mortality of Canaan fir following 2016, the second year following planting.
Whereas all 60 Fraser firs in the 2010 previous planting had died within three years, only 64% of Fraser firs in the no-sulfur plots had died at the end of four years in this Fig. 3. Comparisons of color ratings, height, and cumulative mortality measured from Canaan and Fraser firs planted in acidified and non-acidified plots. Data have been averaged across root dip treatments conducted at the time of planting. The trees were planted as bare-root nursery stock in April, 2015. Data for color ratings and height are given as mean 6 se. Color ratings: 0, brown and dead; 1, almost dead (yellowed and wilting); 2, very yellow; 3, slight yellowing; 4, fully green. experiment. This difference can probably be attributed to a reduction in precipitation. Rainfall through the first two years of the experiment planted in 2015 did not cause frequent, saturated soil conditions conducive to the development of phytophthora root rot to the same extent as in the previous planting in the same field. Rainfall records were assessed from weather stations nearby: T. F. Green Airport, Warwick, RI, 43 km from the field site for May, 2010 to April, 2016, and the East Killingly NEWA (Network for Environmental and Weather Applications, Cornell University) station, 9 km from the field site from May, 2016 to April, 2019. Monthly total precipitation and maximum daily precipitation were compared for the periods from May, 2010 to December 2013 for the earlier experiment, and May 2015 to April 2019 for the present experiment. Trees planted in 2010 experienced 13.9 6 1.3 cm (mean 6 se) of monthly rainfall, with maximum daily precipitation events per month averaging 6.8 6 0.7 cm, and the trees planted in 2015 experienced 10.5 6 0.7 cm (mean 6 se) of monthly rainfall, with maximum daily precipitation events per month averaging 4.1 6 0.4 cm.
Comparison of months with rain events of over 5 cm in daily precipitation are especially striking, with 17 out of 24 months exceeding this rain intensity for the first, versus 3 out of 24 months in the second experiment. There were no precipitation events exceeding 5 cm through all of 2017. Reduction of soil pH through the incorporation of sulfur clearly improved the health of both a susceptible species (Fraser fir) and a less susceptible species (Canaan fir). Richter et al. (2011) found no benefit to preventing phytophthora root rot in plots amended with sulfur prior to planting. However, in that work, the two field sites had initial pH values of 5.4 and 4.8, and so dropping the soil pH to 4 did not create as great a difference between plots as reported in this study. Furthermore, in the Connecticut trial, only modest protection was observed in Fraser fir (the only species planted in the North Carolina study), whereas a much greater degree of protection was provided to Canaan fir. The North Carolina study found that plant mortality of Fraser fir transplants did not co-vary with soil pH in additional research plots to which sulfur had not been added (Richter et al. 2011): once again, focusing on Fraser firs (for which there may be smaller benefits), the smaller variation in pH, and the smaller sample sizes of trees could obscure the potential for soil pH to influence phytophthora disease.
The effects of sulfur incorporation into soil on protecting the roots from Phytophthora infection will require further investigation into the possible mechanisms for the protective effects. Based on a strong inference paradigm, non-mutually exclusive hypotheses that need to be considered are: (1) Phytophthora and other root rot pathogens may be intolerant of low pH soils, in which hydrogen ions may be interfering with production of sporangia or causing mortality in zoospores (Kong, et al. 2009). (2) Elemental sulfur could act directly as a biocide, but this effect was not observed by Cooper and Williams (2004). (3) Conversion of elemental sulfur acidifies soil through its microbial oxidation to sulfuric acid. The H þ produced from this reaction could liberate Ca þþ into soil solution from cation exchange sites, which then could interfere with liberation and movement of zoospores (Maloney et al. 2005). (4) A reduction in soil pH changes mineral availability. Richter et al. (2011) especially noted high tissue concentrations of manganese in Fraser firs planted into plots to which sulfur had been incorporated. Elevated levels of several minerals may protect against infection (Duffy 2007, Evans et al. 2007, Thompson and Huber 2007. (5) Lowered pH may favor microbiota antagonistic to Phytophthora. Evaluating community structure to identify antagonistic microorganisms and the conditions that specifically encourage them could be made difficult by the extraordinary complexity of soil microbiota and the presence of both generalist and specialist antagonists in the soil (McDonald et al. 2007). (6) Soil at higher pH may favor organisms that exacerbate phytophthora root rot, such as plant pathogenic nematodes that could open infection courts for disease, as is seen in several crops (Chen and Rich 1962, LaMondia 2003, Rowe et al. 1985. Although the mechanisms for the beneficial effects are unknown, application of elemental sulfur to reduce the pH of soil appears to be a practical approach that, together with planting Canaan fir, a species less susceptible to this disease, can make production of trees economically viable at the studied site. How readily these results may be extended to other field sites with different soil conditions and other Phytophthora spp. remains to be seen. As with many pest management challenges, this study has demonstrated that combining multiple tactics provides superior results over planting the less susceptible species or incorporation of sulfur alone. Clearly, other practices already known to limit losses from phytophthora root rot, such as improving drainage, planting into raised beds, and possibly the use of organic mulches (Richter et al. 2011) could be combined with planting less susceptible species and soil acidification to further reduce losses from root rots in Christmas tree plantings.