Hydroponic screening of traditional rice varieties in Assam, India to estimate their potential resistance to Al toxicity under P deficiency

Acid soils encompass nearly one-third of the available terrestrial land surface worldwide. Acidic soil is one of the major abiotic constraints for agricultural practices by potentially creating aluminum (Al) toxicity and/or phosphorous (P) deficiency. Assam, being an agricultural state of India, has the majority of its area covered by acidic soils due to the varied terrain in the region. Soil acidification increases the solubility of Al present in the soil from its nontoxic silicate or oxide forms into highly phytotoxic ionic Al (mainly the trivalent cation Al3+). Ionic Al can form complexes with the available phosphorous leading to plant nutrient deficiency. In the present investigation, screening of traditional rice varieties from Assam was conducted for tolerance to combined Al toxicity and P deficiency. Seedlings of 41 rice landraces from various agro-climatic locations were subjected to three different concentrations of Al (0, 50, 100 μM) for 24, 48, and 72 h under P deficiency in static nutrient culture to identify the extent of their resistance to these stressed conditions. Different morpho-physiological parameters (root and shoot lengths, fresh and dry weight yields, chlorophyll and relative water content) were evaluated after stress treatment. All the experiments were conducted in a randomized block design with three replicates. Based on the overall morphological characters, total stress response index (TSRI) was calculated which showed a variation ranging from 18 to 23. Accordingly, the varieties were classified into different groups of resistance. Varieties ‘Moti’ and ‘Baismuthi’ were found to be the least resistant, whereas ‘Holpuna’, ‘Beto’, and ‘Soria Sali’ were identified as most tolerant varieties to Al toxicity under P deficiency. The findings of the present investigation could be exploited for developing promising varieties in future rice breeding programs.


Introduction
Aluminum (Al) is one of the most abundant metals comprising approximately 7% in the Earth's crust. The chemical stability of Al depends upon the pH of the soil. At a neutral pH, Al minerals exist in an almost insoluble state. However, in acidic conditions Al is oxidized to Al 3+ , which is toxic to most of the conventional crops including rice [1][2][3]. As such, Al 3+ binds to soluble phosphorous (P) limiting the plant-available P. Therefore, the adverse effect of an acidic soil on plant growth is strongly related to both the toxicity of Al 3+ and P deficiency.
Very little free Al 3+ in the soil solution causes damage to plant systems. Soluble forms of Al inhibit root growth as well as the development of root hairs. This results in poor nutrient uptake and assimilation and thereby leads to shoot nutrient deficiencies and a reduction of productivity [1,4]. This inhibition of root growth is one of the significant symptoms of Al toxicity, which occurs due to the interaction between Al 3+ ions and root cells and their components [5][6][7].
Plants have a wide range of adaptability to tolerate the soluble form of Al 3+ ; some species can tolerate >1 mg L −1 , whereas most will show an adverse effect even at concentrations <0.5 mg L −1 . A large proportion of soil Al is locked up in soil minerals such as aluminosilicates, with much smaller fractions appearing in soluble forms that are capable of influencing biological systems [8].
Deficiency of P is one of the major limiting factors for rice growth and yield [9]. In upland conditions, P deficiency commonly occurs due to the strong binding affinity of P, which predominantly reacts with Al and iron (Fe) in acidic soils, whereas it reacts with calcium (Ca) in neutral to alkaline conditions and consequently forms partially soluble complexes [10]. As compared to other macronutrients, P is found to be one of the least mobile and available elements in the soil. Phosphorus in the soils occurring in both organic and inorganic forms and P availability is a prime limiting criterion for plant growth and development.
The alarming increase in human population demands the necessity to accelerate agricultural productivity. Hence, it is a challenge for the agricultural community to fulfil the increasing demands for future food supply across the globe. In this context, it is equally important to think about soil health status, which is being impacted due to various abiotic factors caused by climatic changes. Efforts are being made to understand the complex processes of agro-ecosystems and the various interactions governing the sustainability of agricultural lands [11]. However, it is becoming clear that conventional agricultural practices cannot sustain the production base to maintain a healthy plant/ soil system indefinitely and, as such, augment crop productivity. Currently, agronomists depend heavily on chemical fertilizers. In this context, P is the next most limiting essential nutrient after nitrogen (N), restricting crop growth and productivity. In the global context of soil status, most tropical and subtropical soils are acidic in nature and are thus often P deficient [12].
Acid soils are a serious problem in Assam and most of the areas of Northeast India and Southeast Asia, including Myanmar, Bangladesh, and parts of China. The low pH of Assam soils adversely affects crop productivity, notably of rice [13]. As rice is widely cultivated in Assam, it is therefore imperative for plant biologists to identify promising rice varieties that can thrive under Al toxicity and P-deficient soil conditions. With this in mind, the present study was conducted to explore the potential of some traditional rice landraces from Assam and to identify tolerant varieties to be used in future rice breeding programs.
Seeds were surface-sterilized with 0.1% HgCl 2 for 15 minutes and washed threefour times with sterile distilled water to remove the residues of previous treatments. Germination was carried out on paper film and sterile moistened cotton in a growth chamber at 25-28°C for 72 h. Twenty seedlings of almost equal size were transplanted into transparent plastic vessels and allowed to grow in Hoagland's nutrient solution [14] in the growth chamber under white light with a photon flux density of 220 μmol m −2 s −1 (PAR) over a 14-h photoperiod. The nutrient solution was replaced after 5 days. Nine-day old seedlings were subjected to a 500 μM CaCl 2 (pH 4.5) pretreatment for 24 h in order to maintain homeostasis, followed by treatment with AlCl 3 at different concentrations (0, 50, and 100 μM) for 24, 48, and 72 h in a modified Hoagland's solution where NH 4 H 2 PO 4 was replaced by NH 4 Cl to maintain the P-deficient conditions. Plants grown in regular Hoagland's medium without P deficiency and Al stress were considered as the controls. Three biological replicates were maintained and cultures were placed in a randomized block design in the growth chamber.

Measurement of plant growth and biomass
Plant growth was measured in terms of shoot and root length, fresh weights, dry weights, relative water content (RWC), and chlorophyll content. For each treatment, three replicates consisting of 20 seedlings were considered, and from each replicate five plants were chosen randomly for the measurement of shoot and root lengths. Again, three plants from each replicate were randomly selected for measurement of fresh and dry biomass. Then shoot and root tissues were dried at 80°C for 48 h and weighed to obtain dry biomass.
To determine the relative water content (RWC), the fresh weight was initially measured, then the seedlings were placed in distilled H 2 O for 4 h in diffuse light. When a shoot became fully turgid, it was reweighed. The dry weight was taken after drying for 72 h and the shoot RWC was then calculated [15].

Determination of chlorophyll concentration
For chlorophyll a, b, and total chlorophyll concentrations, fresh leaf samples of 300 mg were crushed in mortar and pestle with 5 mL of 80% acetone. The absorbance was measured at 663 nm and 645 nm using an UV-Vis spectrophotometer (Eppendorf Bio-Spectrophotometer Basic-603873) against 80% acetone as reference, and total chlorophyll was calculated by using a standard formula [15].

Determination of total stress response index
Total stress response index (TSRI) was calculated for the maximum stress duration (72 h) by using the established formula with modifications [16]. Calculation was carried out by dividing the average stress from the average control value for each variety at both Al concentrations (50 µM and 100 µM) for all the parameters determined individually. Summation of all the parameters was made to obtain the datasets for both Al concentrations. Finally, the mean tolerance values of both data sets were calculated to determine the TSRI. A dendrogram was generated by Ward's linkage method based on similarity in total tolerance indices.

Statistical analysis
As the experiments were conducted with a completely randomized design, summarized data were calculated as treatment means ±SE for three replicates. Pearson's correlation coefficient analysis was carried out using the MS Excel data sets and statistical significance was considered at p values <0.05. Data were analyzed using the XLSTAT 2017 statistical package [17]. A cluster analysis of the 41 rice varieties was carried out based on the TSRI data.

Morpho-physiological parameters under Al treatment and P deficiency
Shoot and root length. The growth of the rice seedlings in Hoagland's solution under Al treatment and P deficiency was measured in terms of shoot and root lengths and revealed that the growth of most of the varieties was impacted. The shoot length of all the 41 traditional rice varieties ranged from 15. In the case of the roots, lengths ranged from 4.4-11.9 cm at 50 µM Al to 4.7-11.5 cm at 100 µM Al after 24 h. After 48 h, root lengths did not show marked differences at both the Al concentrations used. After 72 h, the root lengths ranged from 4.2 to 11.5 cm at 50 µM Al and from 4.3 to 11.1 cm at 100 µM Al. Variety 'Bhugpuri joha' showed the lowest (4.4 cm) and 'Moinagiri' was characterized by the highest (11.9 cm) root length at 50 µM Al. Varieties 'Kola joha' and 'Moinagiri' were the lowest (4.7 cm) and the highest (11.5 cm), respectively, at 100 µM Al after 24-h exposure. After 48 h, the lowest and highest root lengths at 50 µM Al were found in 'Bhugpuri joha' (4.4 cm) and 'Moinagiri' (11.9 cm), respectively, and at 100 µM Al in 'Sokbonglong' (4.3 cm) and 'Moinagiri' (11.5 cm), respectively. With an increased stress exposure time to 72 h, 'Sokbonglong' showed the lowest (11.5 cm) and 'Moinagiri' the highest (15.1 cm) root lengths at 50 µM Al, whilst at 100 µM Al, 'Sokbonglong' produced the lowest (4.3 cm) and 'Kartik' the highest (11.1 cm) root lengths (Fig. 2).
Root and shoot fresh weights. The root fresh weights ranged from 0.04 to 0.13 g at 50 µM Al and from 0.04 to 0.17 g at 100 µM Al after 24 h of exposure. After 48 h, they ranged from 0.05 to 0.13 g at 50 µM Al and from 0.05 to 0.14 g under 100 µM Al treatment, respectively. After 72 h, they ranged from 0.04 to 0.15 g at both Al concentrations used. After 24 h of treatment, 'Bhugpuri joha' showed the lowest root fresh weights (0.04 g) at both Al concentrations, whereas 'Bora dhan' showed the highest (0.12 g) root fresh weights at 50 µM Al and 'Baismuthi' emerged as tolerant at 100 µM Al (0.17 g). However, after 48 h of stress, 'Bhugpuri joha' had the lowest (0.05 g) and 'Bora dhan' the highest (0.13 g) root fresh weights at 50 µM Al and Fulpakhri the lowest (0.04 g) and 'Bora dhan' the highest (0.13 g) root fresh weights in 100 µM Al. After 72 h exposure, 'Bhugpuri joha' produced the lowest (0.04 g) and 'Rupohi' the highest (0.15 g) root biomass at 50 µM Al. Varieties 'Nalbora' and 'Basful' had the lowest (0.05 g) and the highest (0.15 g) root fresh weights, respectively, at 100 µM Al (Fig. 3).
The fresh weight of shoots ranged from 0.09-0.31 g at 50 µM Al to 0.18-0.31 g at 100 µM Al after 24 h. However, after 48 h they ranged between 0.16 and 0.30 g at 50 µM Al and between 0.18 and 0.38 g in the 100 µM Al treatment. After 72 h, a range of 0.15-0.29 g in 50 µM Al and 0.15-0.34 g in the 100 µM Al treatment was noted. Variety 'Kola bora' produced the lowest shoot fresh weights (0.09 g) at 50 µM Al and 'Kola joha' the lowest (0.18 g) at 100 µM Al. Variety 'Lauguti' showed the highest shoot fresh weights (0.31 g) in both the Al treatments after 24 h. However, after 48 h, 'Bhugpuri joha' showed the lowest (0.16 g) and 'Lauguti' the highest shoot fresh weights (0.30 g) in 50 µM Al. Simultaneously, 'Moti' produced the lowest (0.18 g) and 'Kekua bao' the highest (0.30 g) shoot fresh weights at 100 µM Al. After 72 h of Al exposure, 'Bhugpuri joha' demonstrated the lowest shoot fresh weights (0.15 g) in both the Al treatments used and ' Adoliya' showed the highest (0.29 g) at 50 µM Al. 'Kartik' had the greatest shoot fresh weights (0.34 g) at 100 µM Al (Fig. 4).  Root and shoot dry weights. Root dry weights ranged from 4 to 10 mg at 50 µM Al and from 3 to 17 mg at 100 µM Al after 24 h in the stress treatments. After 48 hours, the dry weights ranged from between 3 and 58 mg at 50 µM Al and between 3 and 50 mg at 100 µM Al and after 72 h, a range of 3-10 mg in 50 µM Al and 3-9 mg in the 100 µM Al concentrations were found. With all the times of stress duration, 'Bhugpuri joha' showed the lowest root dry weights (3 mg) at both the Al concentrations used. 'Bora dhan' had the highest root dry weights (10 mg) at 50 µM Al, whilst 'Rupohi' emerged as tolerant (17 mg) in 100 µM Al after 24 h. However, after 48 h, 'Baismuthi' revealed the highest root dry weights (50 mg) in both Al treatments. After 72 h, 'Bhugpuri joha' demonstrated the lowest (4 mg) and 'Rupohi' and 'Kartik' the highest (10 mg) root dry weights at 50 µM and 100 µM Al, respectively (Fig. 5). After 24 h, the shoot dry weights ranged from 18 to 80 mg in the 50 and 100 µM Al treatments, respectively. After 48 h, they ranged from between 17 and 90 mg at 50 µM Al and between 10 and 70 mg at 100 µM Al. After 72 h, a range of 18-50 mg for 50 µM Al and 19-60 mg for 100 µM Al was noted. With 24-h treatment, varieties 'Lucky' and 'Bhugpuri joha' produced by the lowest shoot dry weights at 50 µM Al and 100 µM Al, respectively. 'Rupohi' was seen to have highest (80 mg) shoot dry weights. However, after 48 h, 'Bhugpuri joha' showed the lowest shoot dry weights (17 mg) at both Al concentrations and ' Anjana' the highest shoot fresh weights (90 mg) at 50 µM Al and 'Kartik' showed the highest shoot dry weights (70 mg) at 100 µM Al. Again, after 72-h stress treatment, 'Bhugpuri joha' showed the lowest shoot dry weights (18 mg) in both the Al concentrations and 'Rupohi' the highest shoot fresh weights (56 mg) at 50 µM Al. Variety ' Anjana' produced the highest shoot dry weights (60 mg) in 100 µM Al (Fig. 6).

Relative water content (RWC).
To understand the effect of Al stress, relative water content (RWC) of the leaves was monitored in both control and treated plants. RWC showed significant differences in both the Al stress regimes. Varieties 'Jahingia' and 'Moti' showed higher RWCs at 24 h, 'Bati Sali' and 'Hung bora' at 48 h, and 'Betguti' and 'Bor joha' showed higher RWCs after 72 h. Varieties 'Nal bora' and 'Prasad bhog' showed the lowest RWCs at 24 h and 48 h, whereas at 72 h, a significant reduction in RWC was found in ' Anjana' at 50 µM Al and in 'Baismuthi' at 100 µM Al (Fig. 7).
Chlorophyll concentration. All the rice varieties tested revealed variations in total chlorophyll content in the Al-treated plants compared to the control. The total chlorophyll concentrations ranged from 5.2 to 23.3 mg mL −1 at 50 µM Al and from 3.9 to 21 mg mL −1 at 100 µM Al after 24 h. After 48 h of treatment, ranges of 3.9-11.8 mg mL −1 at 50 µM Al and 3.8-11.56 mg mL −1 at 100 µM Al were determined. After 72 h, the ranges were 5.8-12.1 mg mL −1 for 50 µM Al and 5.3-10.6 mg mL −1 for 100 µM Al. Among all the rice varieties, ' Anjana' showed a significantly highest total chlorophyll concentration, whereas 'Moti' , 'Maguri bao' , and 'Basful' showed lower chlorophyll concentrations over the respective time frames (Fig. 8).

Total stress response index (TSRI)
All the morpho-physiological parameters were statistically analyzed and based on these results, TSRI was calculated which clearly illustrated the differences in plant resistance to Al toxicity under P deficiency between the rice varieties. Based on its TSRI value, variety 'Holpuna' could be identified as the most resistant, 'Soria Sali' and 'Beto' as moderately resistant, whilst 'Baismuthi' and 'Moti' are the most sensitive (Fig. 9). On the basis of the differences of all the TSRI values, the varieties can be classified into four groups: highly resistant (≥4.0), moderately resistant (3-4), susceptible (2-3), and highly susceptible (≤2.0) to Al toxicity under P deficiency.
Clustering based on morpho-physiological parameters of rice grown under Al toxicity and P deficiency A dendrogram was constructed and a cluster analysis performed based on distance matrices which revealed two major clusters. Cluster I comprised 28 rice varieties and Cluster II 13 varieties. Additionally, Cluster I was further divided into two groups and Level of significance: * p < 0.05 (two-tailed). RL -root length; SL -shoot length; RFW -root fresh weight; RDW -root dry weight; SFW -shoot fresh weight; SDW -shoot dry weight; RWC -relative water content; CHL -chlorophyll concentration.
Cluster II formed two major groups (C, D). Group D could be further divided into three subgroups. The selected susceptible varieties 'Moti' and 'Baismuthi' clustered together revealing their similarity in Al response. Varieties 'Soria sali' and 'Beto' clustered together again suggesting moderate similarity, whereas the highly tolerant 'Holpuna' emerged in a different cluster justifying our conclusions on the basis of morpho-physiological parameters under Al toxicity and P deficiency (Fig. 10).

Discussion
It has been demonstrated in previous works that P supply can ameliorate Al toxicity possibly through precipitation of Al in the rhizosphere. There arises a relative ranking of Al tolerance in plants and the probable cause is due to P and Al interactions which exist in acidic soils. Under P-limiting conditions, it was observed that P-efficient genotypes acquire more P from soils and transport more P to the root tips resulting in an increase in Al tolerance [18]. Adaptation to low-P acidic soils might involve a superior ability to tolerate Al and utilize organic P efficiently. Keeping this in mind,   the present experimental model was designed with modification in composition from the basal Hoagland's nutrient solution. It is necessary to lower the P concentration in the growth medium to enable a conclusive comment on the effect of the applied Al concentration. In Hoagland's medium, NH 4 PO 4 is the P source and therefore, in our experimental growth medium, NH 4 PO 4 was substituted by NH 4 Cl to create P-deficient conditions [19]. Here, the tolerance to different concentrations of Al (50 µM or 100 µM) in the growth medium of the 41 rice cultivars collected from various agroclimatic zones of Assam, India was evaluated and the effect of Al 3+ ions on different growth parameters was determined. Inhibition of root growth parameters such as root length and root fresh or dry weights was prominent in presence of the concentrations of Al employed in the growth medium. The roots showed the classical symptoms of Al toxicity, notably a reduction in root growth and the development of lateral roots. Stunted root growth was observed in almost all the experimental rice varieties under stress exposure and became more apparent with its duration. Root growth was reduced at both the Al concentrations and for all durations, and there was a distinct reduction at 100 μM Al for 72 h. This observed reduced root growth behavior was similar to earlier reports by other authors [20][21][22].
Severe reduction of fresh and dry weight and relative water content of roots and shoots were observed in the Al treatments. A decrease in RWC and chlorophyll content was also observed to some extent. The effect was generally greater at the higher concentration of Al (100 µM). These observations also agree with previous results of other researchers [23][24][25][26][27].
Determination of correlations between various morpho-physiological parameters of rice would be useful to select the best combinations of attributes for experimental rice varieties when screening their resistance to Al stress under P deficiency. Correlations between all the eight parameters measured here were calculated which showed significant interaction (p < 0.05) among the different traits. Shoot and root biomass were found to show a significant positive correlation in the presence of Al 3+ ions in the growth medium.
Based upon our observations, we believe that the stress tolerance index can be considered as a suitable marker for screening of a large number of rice varieties to determine the tolerance to a particular environmental stress. In our experiment, this index was useful to differentiate all the screened rice varieties for their tolerance to Al stress and P-deficient conditions. This finding was also supported by the cluster analysis which was found to agree with earlier reports [16,28].

Conclusion
In this experiment, only a few rice varieties were found to show higher resistance than others when exposed to Al stress under P deficiency. Root fresh weight, dry weight, and relative water content were all significantly decreased in the sensitive varieties. The effect of Al 3+ ions was mostly shown by varietes 'Baismuthi' and 'Moti' . Based on the studies of several parameters, it was shown that variety 'Holpuna' is a comparatively resistant variety, whereas 'Moti' is the most susceptible of the 41 cultivars screened. 'Holpuna' can therefore be recommended for Assam farmers. The screening approach we used has also paved a way for further selection of rice varieties which can be used to study the mechanism of enhanced Al tolerance under P deficiency. It can also be beneficial for breeding programs to produce resistant genotypes or to determine gene regulation patterns, and then for producing a genetically-engineered Al tolerant variety.