Assessment of Selected Chickpea Accessions for Tolerance to Aluminium and Manganese Toxicities in Solution Culture and Pot Experiments

doi:10.21203/rs.3.rs-4813191/v1

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Assessment of Selected Chickpea Accessions for Tolerance to Aluminium and Manganese Toxicities in Solution Culture and Pot Experiments

https://doi.org/10.21203/rs.3.rs-4813191/v1

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A good understanding of existing variability in Mn and Al toxicity tolerance among chickpea accessions is required to tackle ion-induced toxicities through the use of tolerant accessions. This study sought to identify acid-tolerant accessions through hydroponic and pot experiments for promotion and adoption in acidic environments. About 26 accessions were screened in nutrient solutions containing 0, 15, and 60µM Al, and 2 and 150µM Mn concentrations, and in pot experiments of pH levels 4.5 and 6.5. The experiments were conducted in a glasshouse with temperatures averaging 17^OC at night and 25^OC during the day. Split plot designs with Al and Mn concentrations as the main plot factor and accessions as the sub-plot factor and replicated thrice were used. Relative tolerance index (RTI), relative root length (RRL), relative shoot length (RSL), relative root weight (RRW), and relative shoot weight (RSW) were used as indices for tolerance for Al, and RSL, RSW, and symptom scores for Mn. Accessions Saina 1, ICCVs 11514, 11519, 11504, 11316, and 07114 were consistently tolerant or moderately tolerant across all indices at 15µM Al. Chania 1, ICCVs 03305, 93954, 07313, 96329, and 97110 were sensitive. At 60µM Al, most accessions were sensitive across all indices. Pot RRL results correlated significantly (r = 0.8) with those from the hydroponic experiments at 15µM Al, with ICCV 11504, ICCV 11514, Saina 1, ICCV 07114, Chania 2, ICCV 11316, ICCV 00108, K036, Leldet 068, and ICCV 10515 being moderately tolerant. In screening for tolerance to Mn, ICCV 07313, 07101, 97128, 11514, Chania 2, and Saina 1 were tolerant. Pot RSL values correlated weakly (r = 0.1) with those of hydroponic experiments. In conclusion, ICCV 11514 and Saina 1 emerged as the accessions with moderate tolerance to soil acidic conditions

Al toxicity

deficiency symptoms

hydroponic

Mn toxicity

moderately tolerant

tolerant

Chickpea cultivation, like that of most other legumes, is limited in acid soils. Currently, the crop is recommended for soils with a pH ranging between 6 and 7 [1]. The occurrence of Aluminium (Al) and manganese Mn toxicities, and phosphorus deficiency constitutes the main limiting factors for crop production in acid soils [2]. Apart from reduced yields, other desired chickpea qualities such as nitrogen fixation and the nature of mineral nutrition are also affected [3]. While the application of lime and p-fertilizers can remedy acidity problems, such remedies are seldom cost-effective, and in the case of lime, ineffective at lower soil horizons [4]. This leaves the use of tolerant accessions as the most effective solution in chickpea cultivation. In Kenya, approximately 20% of land under cultivation is acidic [5]. While these soils occupy a small percentage of total arable lands, they are common in the high-potential areas that serve as the country’s food basket [6]. Additionally, excessive use of ammonium fertilizers and acid rains have exacerbated the problem in previously acid-free soils, and hence their unsuitability for chickpea production [7].

Low fertility in acid soils is attributed to many factors that include Al toxicity, Mn toxicity, and deficiencies of other nutrients such as P, Ca, Mg, and Zn [8]. However, Al toxicity is the most important constraint to crop production in such soils [9]. Al occurs naturally in soil solution in forms that are not harmful to biological processes in living things [10]. However, when soil solution pH drops below 5.5, non-toxic forms of Al solubilize into toxic forms such as Al³⁺, which interfere with cellular processes upon interaction with plant roots [9].

Al toxicity in plants occurs through inhibition of root growth, disturbances to cytoplasm calcium, inducement of oxidative stress, and nutrient imbalances in the cell wall and plasma membranes [11]. Root exposure to Al inhibits growth and consequently the plant’s nutrient uptake [12]. However, different plant species display varying levels of tolerance to Al toxicity [13]. Additionally, the variance in tolerance levels has also been observed among varieties belonging to similar plant species [14]. Chickpea accessions have also been found to display variable levels of Al tolerance [15]. Singh and Choudhary [16] demonstrated Al tolerance among chickpeas to be an inheritable trait and hence the possibility of breeding tolerant accessions through backcross breeding programs. Existing genetic diversity among both domesticated and wild chickpea accessions present important sources of tolerance to Al toxicity among other stressors [17].

Besides Al, Mn element is another common constraint limiting crop production in acid soils. The element is essential for plant growth as it plays important roles in protein structure and photosynthesis [18]. Its deficiency affects water photolysis in photosynthesis leading to stunted plant growth and development. However, its excessive presence is also toxic to plants under certain soil and climatic conditions [19]. At soil pH levels below 5.5, the concentration of Mn ions increases and consequently becomes toxic to plants. According to Khabaz-Saberi et al. [20], waterlogging and high soil temperatures exacerbate Mn toxicity even at pH levels as high as 6. Toxic levels of Mn can also be observed in soils at high pH levels if the parent material contains high levels of total Mn.

Mn toxicity to plants occurs in several ways. Lavres Junior et al. [21] reported alteration of enzymatic activities in the cell cytoplasm, and disturbances in the uptake, use, and redistribution of other nutrients due to the presence of excessive concentrations of Mn in plant tissues. Another study by Santos et al. [18] found a decrease in shoot biomass among soybean plants exposed to excessive concentrations of Mn, an observation attributed to reduced stomata conductance and lower CO₂ assimilation rates. Mn toxicity in plant tissues results in the degradation of compounds such as proteins, nucleic acids, and carbohydrates, hence causing cell death [22]. Mn toxicity is also believed to reduce photosynthesis, an observation that Weng et al. [23] attributed to the degradation of photosynthetic material in cell chloroplast.

Mn toxicity is very detrimental to chickpea growth and development [24]. Upon exposure, chickpeas display symptoms such as reduced leaf size, yellowing of serrated margins of leaves, and reduced shoot growth [25]. Additionally, absorption of nutrients such as Ca, Mg, and P is greatly limited under Mn stress [16, 24]. However, chickpea response to Mn toxicity and acidic soil conditions varies across different accessions [26]. This provides an opportunity for development of Mn tolerant accessions. Al and Mn toxicity to plants in acid soil can be corrected through the use of tolerant accessions. Identification of such tolerant accessions can be done through field experiments and in solution culture [27]. Field experiments, however, are very costly and take a longer time when many genotypes are being tested [28]. Screening in solution culture is a quick and effective method that discriminates between tolerant and sensitive plant genotypes. It is reliable over field methods due to the variation in soil conditions even over small areas in acidic soils that make the latter more difficult to undertake [29]. Studies have shown a positive correlation between results obtained through solution culture screening and those obtained in the field [28]. This study, therefore, was aimed at screening selected chickpea accessions for tolerance to Al and Mn toxicities, and to identify promising lines for future field trials.

All experiments were conducted in a glasshouse at the University of Nairobi, Upper Kabete Campus. Temperatures in the glasshouse averaged 17 ◦C at night and 25 ◦C during the day. Twenty-nine chickpea accessions (13 Kabuli types and 16 desi) were selected for use in these experiments (Table 1). Seven accessions were obtained from farmers in regions identified to have acidic soils in Kenya including parts of Bomet, Laikipia, Embu, and Machakos Counties. Another 22 selected for prior exposure to acid soils were sourced from the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Nairobi. The accessions were screened for tolerance to Mn and Al toxicities in solution culture (hydroponically) and in pot experiments in a glasshouse with cowpea used as the control.

3.1 Plant Materials and germination tests

Seed germination tests were conducted for all the accessions before the start of the experiments. A majority of the seeds had germination rates at or above 50 percent. Seeds with germination below 50% were replaced. Three accessions that displayed zero germination rates (ICCV 10516, ICCV 95311, and Kenya Ngara Local) could not be replaced in time for the start of the experiment, reducing the total number of varieties screened to 26 (Table 1).

Table 1

Description of plant material used in the study
	Source/Release name	Regen Year (Year of breeding/introduced)	ACCENAME (Breeding identifier)	Country of origin	Basic descriptor/seed color
1	ICRISAT/_	Not released	ICCV 11316	India	Kabuli/cream
2	ICRISAT/_	Not released	ICCV 93954	India	Kabuli/cream
3	ICRISAT/_	Not released	ICCV 97125	India	Desi/brown
4	ICRISAT/_	Not released	ICCV 11504	India	Desi/brown
5	ICRISAT/_	Not released	ICCV 11514	India	Desi/brown
6	ICRISAT/_	Not released	ICCV 11519	India	Desi/brown
7	ICRISAT/_	Not released	ICCV 10515	India	Desi/brown
8	ICRISAT/_	Not released	ICCV 10516	India	Desi/brown
9	ICRISAT/Chania 1 Egerton	2011	ICCV 97105	India	Desi/brown
10	ICRISAT/Chania 2 Egerton	2014	ICCV 92944	India	Desi/brown
11	ICRISAT/Chania 3 Egerton	2014	ICCV 97126	India	Desi/brown
12	ICRISAT/_	Not released	ICCV 97128	India	Desi/brown
13	ICRISAT/_	Not released	ICCV 03305	India	Kabuli/cream
14	ICRISAT/_	Not released	ICCV 07313	India	Kabuli/cream
15	ICRISAT/_	Not released	ICCV 07114	India	Desi/brown
16	ICRISAT/_	Not released	ICCV 07101	India	Desi/brown
17	ICRISAT/_	Not released	ICCV 08107	India	Desi/brown
18	ICRISAT/_	Not released	ICCV 97110	India	Desi/brown
19	ICRISAT/Saina 1 /KALRO/EGERTON	2011	ICCV 95423	India	Kabuli/cream
20	ICRISAT/_	Not released	ICCV 95311	India	Kabuli/cream
21	ICRISAT/_	Not released	K036	Kenya	Kabuli/cream
22	ICRISAT/LELDET 068	2014	ICCV 00305	India	Kabuli/cream
23	Farmer’s field/ndaria	Introduced to farmers by ICRISAT in the 1970s	-	Kenya. Sourced from Nanyuki (0^o10’48’’S 36^o89’67’’E)	Kabuli/cream
24	Farmer’s field/Kenya ngara local	Introduced to farmers by ICRISAT in the 1970s	-	Kenya. Sourced from Naro moru (0^o11’23’’N 37^o42’27’’E)	Kabuli/cream
25	Farmer’s field/Israel	Sourced from Israel in 2015	-	Israel. Sourced from Athi river (1^o55’62’’S 37^o10’13’’E)	Kabuli/cream
26	Farmer’s field/ Saina ndogo	Introduced to farmers by ICRISAT in the 1970s	-	Kenya. Sourced from Embu (1^o12’02.5’’S 37^o33’11’’E)	Desi/brown
27	Farmer’s field /Saina kubwa	Introduced to farmers by ICRISAT in the 1970s	-	Kenya. Sourced from Matuu (1^o20’69.3’’S 37^o54’90’’E)	Kabuli/cream
28	Farmer’s field/Nubirbi	2020	ICCV 96329	Kenya. Sourced from Longisa (0^o54’37.9’’S 35^o23’34.7’’E)	Kabuli/cream
29	Farmer’s Field/Nubirbi	2010	ICCV 00108	Kenya	Desi/brown

Source: International Crops Research Institute for the Semi-Arid Tropics - write in full

3.2 Experiment 1: Screening for tolerance to Al toxicity

All accessions were labeled by assigning numbers 1–26 for ease of processing and data entry. Seeds were scarified by removing a tiny piece of the coat using a scalpel. Scarification was done by holding a seed between the thumb and the first finger and a tiny piece of its coat chopped off. The seeds were then disinfected using 4% sodium hypochlorite for five minutes and rinsed using running tap water for an additional 5 minutes. Sterilized seeds were transferred to glass petri dishes with bottoms covered with paper towels for moisture retention. Each petri dish carried 15 seeds. About 20 ml of tap water was added to each petri dish and then transferred to a growth chamber for pre-germination.

The seeds were pre-germinated for four days before sowing. Temperatures in the growth chamber were maintained at 26 degrees centigrade. After four days of pre-germination, the petri dishes were removed from the germination chamber and three seedlings per accession were selected for sowing in a dilute nutrient solution with three treatments supplied as AlCl₃.6H₂O (0, 15, and 60µM). The nutrient solution contained CaCl₂.2H₂O, 400µM; MgCl₂.6H₂O, 250µM; NH₄NO₃, 40µM; (NH₄)2SO₄, 10µM; KNO₃, 650µM; H₃BO₃, 23µM; MnCl₂.4H₂O, 9µM; Na₂MoO₄.2H₂O, 0.1µM; ZnSO₄.7H₂O, 0.8µM; CuSO₄.5H₂O, 0.3µM; Na₂HPO₄, 5µM; C₁₀H₁₄Na₂O₈.2H₂O, 20µM; and FeCl₃.6H₂O, 20µM [17]

Three seedlings were transplanted for an accession per treatment. A split plot design with three replicates was used with the Al concentrations as the main plot factor and accessions as the sub-plot factor. The seedlings were inserted into holes on a styrofoam material with polyurethane foam wrapped around each seed (Fig. 1). The polyurethane material held seedlings in place without impending root and shoot growth while the styrofoam material ensured that the seedlings floated on the nutrient solution. The styrofoam sheets with seedlings were then transferred to a glasshouse and placed on the containers with the nutrient solution. The containers measured 35 cm × 29 cm × 21 cm and each carried a maximum of 33 seedlings. Three containers were used for each treatment. Accession positions in the styrofoam sheet and that of containers in the glasshouse were assigned randomly.

The nutrient solution was constantly aerated by bubbling air through it using a compressor pump. The pH of the solution was maintained at 4.2 throughout the experiment by adding either 0.1M HCl or 0.1M NaOH. pH measurements were taken daily and adjusted to 4.2 when necessary. Additionally, the nutrient solution together with their respective treatments were replaced every other day, and a sample solution collected before and after each solution change. The experiment was terminated after 10 days.

3.2.1 Accession Sampling, Measurements, and Calculation of Indices

Harvested seedlings were rinsed using distilled water to wash away surface contaminants. Thereafter, they were separated into roots and the shoots and the longest root length (LLR) was measured using a ruler (mm). The shoot length was also measured from its point of separation from the root to the tip of the longest leaf/branch when straightened. Additionally, fresh weights were taken using a weighing scale. The plant samples were packed in khaki bags and transferred to the laboratory for further nutrient analysis.

Relative tolerance index (RTI) (LLR treated/LLR mean control) and relative root length (RRL) (net root length mean treated/net root length mean control) were used as indices of root tolerance for Al. The RTI was calculated by dividing the LLR of the treated plants by those of the control. For RRL, the average net root length of the treated plants was divided by the average net root length of the control. Relative shoot lengths (RSL) (net shoot length treated/ net shoot length control) and relative root dry weights (RRW) (mean root dry weight treated/ mean root dry weight control) were also calculated. The chickpea accessions were classified as tolerant, moderately tolerant and sensitive, when the RRL, RTI, RSL, and RRW values were: > 70 = tolerant; <=70 and > 50 = Moderately tolerant and < = 50% = Sensitive.

3.3 Experiment 2: Screening for tolerance to Mn toxicity

The same seed preparation procedures and experimental design and setup used for the Al experiment (3.2) were followed. The basal nutrient solution used in this experiment contained: NH₄NO₃, 100 µM; Ca(NO₃)₂4H₂O, 450 µM; MgSO₄7H₂O, 200 µM; K2SO4, 350 µM; NH4H2PO4, 60 µM; CuSO4H2O, 0.2 µM; ZnSO47H2O, 0.6 µM; Na₂MoO₄2H₂O, 0.1 µM; H₃BO₃, 23 µM; FeCl₃.6H₂O, 20 µM; C₁₀H₁₄Na₂O₈.2H₂O, 20 µM; and MnSO4.H2O, 2 µM. 5 ml of MnSO₄.H₂O (sol 7) was used to provide Mn treatments (150 µM) [25].

The pH of the solution was maintained at 5.2 throughout the experiment by adding either 0.1M HCl or 0.1 M NaOH. pH measurements were taken daily and adjusted to 5.2 when necessary. The nutrient solution was replaced weekly. When replacing the solution, the styrofoam sheet would be carefully lifted from the container to minimize disturbance to the growing seedlings. A sample of the solution to be used for laboratory analysis was then taken using a 200 ml bottle before discarding the contents of the container. The styrofoam was then placed back in position and the same process was repeated for all the containers.

The chickpeas initially grew in the basal nutrient solution treated with 2µM MnSO₄.H₂O (control). After 12 days of growth, initial shoot length measurements were taken and treatments were introduced. Half the containers were supplied with 5 ml of 0.3M/L MnSO₄.H₂O (150µM Mn) while the rest retained the control, getting 2.5ml of 0.008M/L MnSO₄.H₂O (2µM Mn). Subsequent nutrient solution changes maintained the treatments and the seedlings were sampled after 28 days of growth.

3.3.1 Accession Sampling, Measurements, and Symptom Scoring

The final shoot length and weight measurements were taken after 28 days of growth and RSL and RSW calculated. Chickpea seedlings were also scored for toxicity symptoms on the 19th and 28th days of the experiment to gauge the effects of the Mn treatments introduced on the chickpea seedlings. The scores were taken using a scorecard ranging from 0 (no symptoms) to 5 (very sensitive) (Fig. 2). The presence and extent of yellowing and brown necrotic spots on leaves, leaf size, and the overall health of the plant were considered when scoring.

3.4 Experiment 3: Screening for tolerance to soil acidity in pot experiments

This experiment was laid out in a split-plot design with the soil pH level as the main plot factor and accessions as the sub-plot factor, and replicated thrice. A total of 26 accessions and a control (cowpea) were grown in soils with different pH levels (4.5 and 6.5). About 2.5 kg of the soil (pH 4.5) sourced from an area known to have acidic soils (Kericho) was prepared from a composite sample and put into 27 plastic pots (one for each accession) while shaking to ensure a uniform bulk density (Fig. 3). The pots measured 25 cm (diameter) by 30 cm (depth). Another 27 pots (control) were supplied with 2.5kg of the same soil with pH adjusted to 6.5 using lime at a rate of 4t/ha as recommended by Kanyanjua et al. [30]. The pots had four drainage perforations at the bottom measuring 1cm in diameter, and each placed on wider plastic plates.

Seed preparation procedures used for the Al and Mn experiments were followed before sowing in the pots at a depth of 1cm and thinned to three (3) after 4 days of growth. Seed sowing rates were determined through pre-germination tests. Field moisture capacity was maintained in all pots throughout the experiment by pouring water on the plates and allowing the soil in the pots to absorb moisture through capillarity. Pot position in the glasshouse was assigned at random.

3.4.1 Accession Sampling, Measurements, and Calculation of Indices

Data collection was done 21 days after sowing. The pots were turned upside down and their contents gently nudged out to ensure minimal disturbance of the plant root structure. The soil was then carefully separated from the accession roots and measurements were taken. Root lengths, shoot lengths, and their fresh weights were measured using a ruler and weighing balance respectively. The measurements were then used to calculate RTI, RRL, RSL, and RRW.

3.5 Plant Analysis

Harvested plant samples from all three experiments were prepared for analysis at the University of Nairobi Soil Chemistry laboratory for nutrient composition. The samples were separated into roots and shoots and oven-dried for 72 hours. Dry samples were digested using a mixture of sulphuric acid, lithium sulphate, and selenium powder [31]. Root and shoot phosphorus contents were determined using methods described by Anderson and Ingram [32]. Aluminium (Al), Calcium (Ca), magnesium (Mg), Manganese (Mn), Iron (Fe), and Zinc (Zn) contents were estimated using an atomic absorption spectrophotometer. The methods are described by Baker [33].

3.6 Statistical analysis

Data obtained from the experiments was subjected to a two-way analysis of variance (ANOVA) to quantify the significance of the treatments. Fisher’s least significant differences (LSD) at P = 0.05 were calculated to show differences in means. Analysis was done using SPSS 13.0 statistical software (SPSS Inc., Chicago, IL, USA) and R-GUI.

4.1 Screening for Tolerance to Aluminium Toxicity

4.1.1 Relative Tolerance Index

The effects of treatments (p < 0.01) and accessions (p < 0.01) on root length were significant, while that of their interaction was not (p = 1). RTI values at 15µM Al ranged between 38.5 and 62% with eleven accessions moderately tolerant and the rest sensitive (Fig. 4). Saina 1 posted the highest RTI of 62%. Other moderately tolerant accessions included ICCV 08107 (61.7%), Saina Kubwa (58.4%), ICCV 11514 (58.1%), 11316 (55.6%), 11504 (54.8%), 11519 (52.6%), Saina Ndogo (52.1%), 07101 (51.1%), 07114 (50.9%), and 00108 (50.4%). The most sensitive accession was Chania 1 with an RTI score of 38.5%. Israel (40.8%), ICCV 03305 (43.5%), 97128 (44.5%), and 93954 (44.5%) were also very sensitive to Al at 15µM. At 60µM Al concentration, RTI values were markedly lower than those at 15µM and ranged between 21 and 50%. All the accessions were sensitive to Al at this treatment level. ICCV 08107 had the highest RTI at 50%. Other accessions with relatively high RTIs included Saina 1 (49.1%), ICCV 07101 (48.1%), and Saina Kubwa (46.7%). The least RTI was posted by K036 at 21%. Other accessions whose root growths were severely hampered were Chania 1 (30.3%), ICCV 11519 (31.5%), and ICCV 07114 (32.1%). RTI values of all accessions at both treatment levels were less than those of cowpea (control).

4.1.2 Relative Change in Root Length

RRL ranged between 35.3 and 69.4% at 15µM Al with ten accessions moderately tolerant and seventeen sensitive (Fig. 5). Among the moderately tolerant accessions, ICCV 11514 posted the highest RRL of 69.4%. Other moderately tolerant accessions included ICCV 11316 (63.8%), K036 (63.8%), Saina 1 (61.1%), ICCV 11504 (58.8%), Chania 2 (57.5%), ICCV 11519 (57%), 10515 (56.7%), 07114 (53.3%) and 00108 (51.3%). RRL values at 60µM Al concentration were much lower than those of 15µM, ranging between 3.3 and 39.1%. All accessions were sensitive. ICCV 10515 (39.1%), 11504 (33.5%), and 11514 (30.8%) posted the highest RRL values. Accessions ICCV 08107 (3.3%), Chania 1 (3.6%), and ICCV 00108 (4.6%) posted the least RRL values. RRL values of all accessions at both treatment levels were less than those of cowpea (control).

4.1.3 Relative Shoot Length

The effects of treatments (p < 0.01) and accessions (p = 0.045) on shoot lengths were significant, while that of their interaction (p = 1) was not. RSL values at 15µM Al ranged between 34.2% and 63.5% with fourteen accessions moderately tolerant and the rest being sensitive (Fig. 6). Saina 1 (63.5%), ICCV 11504 (63.3%), 10515 (59.7%), K036 (59.4%), and 11519 (59.2%) were moderately tolerant. ICCV 97125 (34.2%), Chania 3 (39.2%), Chania 1 (41.9%), and ICCV 07313 (42.9%) were sensitive. RSL correlated strongly and positively with RRL (r = 0.8), signifying that both root and shoot growth were inhibited by 15µM Al. At 60 µM Al, RSL values ranged between 24.9% and 56.7%. While the range of values slightly increased when compared to the 15µM Al treatment level, shoot lengths decreased in general. Only four accessions were moderately tolerant and included Saina 1 (56.7%), ICCV 10515 (52.7%), ICCV 11519 (51.5%), and K036 (50.9%). The most sensitive accessions included ICCV 07313 (24.9%), ICCV 96329 (26.7%), and ICCV 97125 (27.2%).

4.1.4 Relative Root Weight

The effects of treatments (p < 0.01) and accessions (p < 0.01) on root weights were significant, while that of their interaction was not (p = 1.0). RRW values 15µM Al ranged between 35.6% and 74.7% (Fig. 7). Fourteen accessions posted RRWs > 50 while the rest were below 50%. Accessions with the highest RRWs included ICCV 11519 (74.7%) Saina 1 (60.8%), ICCV 11504 (60.8%), and Saina Ndogo (60.6%). ICCV 07313 (35.6%), ICCV 93954 (40%), and ICCV 97125 (40.9%) posted the least RRW values. At 60µM Al, RRWs ranged between 24.3% and 60.5%. RRW at 15µM correlated strongly and positively with RRL (r = 0.7) and RSL (r = 0.8) at the same treatment level.

4.1.5 Relative Shoot Weights

The effects of treatments (p < 0.01) and accessions (p < 0.01) on shoot weights were significant, while that of their interaction (p = 1.0) was not. RSL values at 15µM Al ranged between 33.5% and 71.4% (Fig. 8). ICCV 11504 (71.4%) ICCV 11519 (61.7%) and Ndaria (60%) posted the highest RSW values while ICCV 03305 (33.5%), Chania 3 (37.1%), and ICCV 07101 (37.7%) posted the lowest. At 60µM Al, shoot weights markedly dropped, ranging between 29.1 and 65.1%. RSW values for all accessions at both treatment levels were less than those of the control (Cowpea).

4.2 Screening for Tolerance to Manganese Toxicity

4.2.1 Symptom Scores

Most accessions started displaying very mild to mild symptoms a few days after exposure to 150µM Mn with marginal leaf chlorosis a common occurrence. After fourteen days, the symptoms had worsened. At this stage, most accessions displayed either mild or moderate symptoms with the marginal leaf chlorosis extending inwards towards the midrib. Reduced leaf sizes and curling were also observed among accessions with more severe symptoms. However, no leaflet droppings were observed except among cowpeas (control).

Manganese treatments (p < 0.01) and accessions (p < 0.01) had a significant effect on symptom scores while their interaction (p = 1) was insignificant. After sixteen days of exposure to the treatments, average symptom scores at 150µM Mn ranged between 1.67 and 4.0 (Fig. 9). Three accessions, ICCV 11519, Saina 1, and ICCV 07313 emerged with the highest average scores of 1.67, 1.67 and 1.77 respectively. Twenty accessions were tolerant, five accessions were moderately tolerant and one, Leldet 068, was sensitive. Cowpeas, a legume known to be Mn sensitive and used as the control, were severely affected, posting a score of 4.52 with severe necrotic spotting and leaf drops observed. No symptoms were observed among seedlings grown under 2µM Mn (control treatment).

4.2.2 Relative shoot length (RSL) and Relative shoot weights (RSW)

Manganese treatments (p < 0.01) and varieties (p < 0.01) had significant effects on shoot lengths. Interaction between the two variables was, however, insignificant (p = 1.0). RSL ranged between 30% and 91% (Fig. 10). Six accessions were tolerant, fourteen were moderately tolerant, and six were sensitive. ICCV 07313 posted the highest RSL score of 91%. Other tolerant accessions included ICCV 07101 (84%), ICCV 97128 (82%), ICCV 11514 (79%), Chania 2 (73%), and Saina 1 (70%). ICCV 10515 (30%), ICCV 07114 (35%), Ndaria (38%), Chania 1 (45%), and Saina Kubwa (47%) were the most sensitive accessions. RSL correlated positively with symptom scores (r = 0.55).

Manganese treatments and varieties had significant effects on shoot weights (p = 0.01). However, their interaction was insignificant (p = 0.01). Relative shoot weights ranged between 33.4 and 92.7% (Fig. 11). ICCV 00108, ICCV 97110, Saina Ndogo, ICCV 97125, ICCV 11514, and ICCV 97128 posted the highest RSW values while ICCV 07114, Ndaria, Leldet 068, and Saina 1 posted the least.

4.3 Screening for Acidity Tolerance Through Pot Experiments

4.3.1 Relative Tolerance Index and Relative Root Length

The effects of treatments (p < 0.01) and accessions (p < 0.01) on pot root lengths were significant, while that of their interaction (p = 1) was not. RTI after 14 days of growth ranged between 44.2 and 72.1% (Fig. 12). Four accessions were tolerant, nineteen were moderately tolerant, and only three sensitive. ICCV 11514 was the most tolerant accession with an RTI of 72.1%. Other tolerant accessions included K036, ICCV 10515, and Saina1. RRL ranged between 28.3 and 59.6%. ICCV 11504 (59.5%), ICCV 11514 (59.3%), Saina 1 (58.9%), ICCV 07114 (57.2%), Chania 2 (56.9%), ICCV 11316 (56.8%), ICCV 00108 (55.8%), K036 (52.4%), Leldet 068 (52.2%), and ICCV 10515 (51.0%) were moderately tolerant. The rest of the accessions were sensitive, with Chania 3 (28.3%), Chania 1 (29.4%), and ICCV 03305 (34.5%) being the most sensitive. Pot RRL results correlated significantly (r = 0.8) with those from the hydroponic experiments at 15µM Al. Saina 1, ICCV 11504, ICCV 11514, K036, ICCV 11316, Chania 2, ICCV 07114, and ICCV 00108 emerged as the standout tolerant accessions in both experiments.

4.3.2 Relative Shoot Length

The effects of treatments (p < 0.01) and accessions (p < 0.01) on pot shoot lengths were significant, while that of their interaction (p = 1.0) was not. RSL values for the accessions ranged between 42.9 and 81.2% (Fig. 13). Four accessions were tolerant, including ICCV 11514 (81.2%), ICCV 00108 (76.7%), K036 (76%) and ICCV 11316 (74.1%). Sixteen accessions were moderately tolerant and six sensitive. Chania 3 (42.9%), ICCV 97125 (45.9%), Chania 1 (45.9%), and Saina 1 (46.1%) were the most sensitive accessions.

4.3.3 Relative Root and Shoot Weights

The effects of treatments (p < 0.01) and accessions (p < 0.01) on root and shoot weights were significant, while that of their interaction (p = 1.0) was not. RRW values ranged between 38.7 and 69.5% (Fig. 14). ICCV 11504 (69.5%), Chania 2 (64.8%), and K036 (64.1%) were the best-performing accessions. Shoot weights were not as affected as the root weights by the soil acidity, with RSW values ranging between 47.2 and 76.3%. ICCV 07313 (91.1%), ICCV 08107 (76.3%), ICCV 93954 (74.1%), Israel (70.7%), and Chania 1 (70.2%) posted the highest RSW values.

5.1 Screening for Tolerance to Aluminium Toxicity

A variation in Al toxicity tolerance was observed among the chickpea accessions screened. At 15µM Al, accessions Saina 1, ICCV 11514, ICCV 11316, and ICCV 07114 were consistently moderately tolerant across all indices. Accession ICCV 11519 was tolerant based on RRW, and moderately tolerant across all other indices while ICCV 11504 was tolerant based on RSL and also moderately tolerant across all other indices. Accessions Chania 1, ICCV 03305, ICCV 93954, ICCV 07313, ICCV 96329, and ICCV 97110, on the other hand, were sensitive across all indices. At 60µM Al, all accessions were sensitive across all indices except Saina 1, K036, ICCV 11519, and ICCV 10515 which were moderately tolerant based on RSL, ICCV 11316, 10515, 11514, 07114, 97128, and 11519 based on RRW, and ICCV 11504, 11519 and K036 based on RSW. The differing responses to Al toxicity can be attributed to the genetic variability present among the accessions. Similarly, Negusse et al. [34] reported varied accession response to Al toxicity in a study to identify acid-tolerant chickpea accessions in Ethiopia. Another study by Vance et al. [17] found domestic and wild chickpea cultivars to display different tolerance levels in solution culture screening. Genotypic variation in tolerance to Al toxicity has also been reported in other crops such as maize, wheat, and sunflowers [20, 35, 36]. Al treatments selected for use in this experiment were 15 and 60µM, with the hypothesis that tolerant accessions would be observed at 15 µM, and the extremely tolerant at 60 µM [17].

In this experiment, both root and shoot growth indices were used to determine accession tolerance to Al toxicity with largely similar results achieved. At 15µM Al, accession scores ranged between 38.5 and 62% for RTI, 35.3 and 69.4% for RRL, 34.2 and 63.5% for RSL, 35.6 and 74.7% for RRW, and 33.5 and 71.4% for RSL. A similar trend was observed at 60µM Al, signifying that both root and shoot growth were inhibited by Al toxicity. Additionally, strong and positive correlations between root and shoot indices were observed where RSL correlated strongly and positively with RRL (r = 0.8), while RRW also correlated strongly and positively with RRL (r = 0.7) and RSL (r = 0.8) at 15µM Al. These results, therefore, signify that both shoot and root indices can be used to determine chickpea tolerance levels to Al toxicity, in agreement with other studies. Vance et al. [17], for example, determined RRL to be the most accurate discriminator of Al tolerance in chickpea screening. Root length has also been used to discriminate between Al-sensitive and Al-tolerant accessions in barley, soya beans, and dolichos beans among others [37–39].

The level of tolerance observed at 15µM was not maintained at 60µM. At 15µM Al, ICCV 11514, ICCV 11316, K036, Saina 1, ICCV 11504, ICCV 11519, and ICCV 07114 were moderately tolerant across all indices. At 60 µM Al, all these accessions were sensitive based on RRL and RTI. Saina 1 and ICCV 11504 were moderately tolerant based on RSL and RSW respectively; ICCV 11316, 11514, and 07114 based on RRW; and ICCV 11519 across all three indices. Accession decrease in tolerance with increased Al concentration is attributed to the increase in Al³⁺ in solution culture that severely damaged chickpea roots and hence impeding the absorption of nutrients and water. Inconsistent displays of tolerance in chickpea at different Al concentrations levels was also reported by Vance et al. [17] when screening wild chickpea accessions for tolerance to Al toxicity.

5.2 Screening for Tolerance to Manganese Toxicity

At 150µM Mn, reduced shoot growth, yellowing of leaf tips and serrated margins, and reduced leaf sizes was observed. These symptoms worsened over time and varied among the accessions resulting in scores ranging between 1.67 and 4. These observations are attributed to accumulation of Mn ions in plant roots and shoots that reduced nutrient uptake and translocation. Among cowpeas the symptoms were more severe and leaf drops were observed, an indication of better tolerance to Mn toxicity among the chickpea accessions. The Mn toxicity symptoms in chickpeas observed in this study were also reported by Ahlawat [40] and El-Jaoual and Cox [41], who observed marginal chlorosis and necrosis of leaves, dark reddish brown leaf spots, reduced root growth, and distorted small-sized leaves.

Symptom scores correlated weakly with RSL (R = 0.55). Based on symptom scores, 20 accessions were tolerant, 5 moderately tolerant, and 1 sensitive while for RSL, 6 accessions were tolerant, 14 moderately tolerant and 6 sensitive. The weak correlation is attributed to the effect of environmental and other external factors that may skew symptom scoring. In this case, the presence of ideal environmental conditions for crop growth in the glasshouse may have masked the effects of Mn toxicity and therefore reduced the severity of the symptoms observed. These findings agree with those of Fernando and Lynch [19] and Pradeep et al. [25] who reported that accession genetics and environmental factors such as light intensity and temperature may have an impact on symptom scoring. As such, accession symptom scores and corresponding shoot growth may not be closely related [25]. Therefore, incorporating plant growth parameters alongside symptom scoring in screening chickpeas for Mn tolerance is necessary.

Accession variance in tolerance to Mn toxicity was observed. Based on symptom scores, all accessions were tolerant except ICCV 07114, Chania 1, ICCV 10515, ICCV 03305, and Israel which were moderately tolerant, and Leldet which was sensitive. Accessions ICCV 97128 and ICCV 11514 were tolerant while ICCV 07114 and Ndaria were sensitive based on RSL and RSW. Accession variation in tolerance to Mn is attributed to the genetic diversity present among them. These results agree with those of Pradeep et al. [25] who found differing responses to Mn toxicity at different treatment levels among prominent chickpea accessions in Australia. Studies on other crops have also documented genotypic differences in tolerance to Mn. Khabaz-Saberi et al. [20], for example, reported varying levels of Mn toxicity tolerance among different wheat genotypes.

Reduced shoot lengths and shoot biomass were observed among the treated plants. This is attributed to reduced photosynthesis resulting from Mn toxicity, an occurrence thought to result from decreased availability of oxygen during periods of metal stress in plants [42]. The reduction can also be attributed to damaged cell metabolism and cell death induced by the intake of excess Mn. Mn toxicity degrades lipids, carbohydrates, proteins, and nucleic acids, damaging cell metabolism, and even cell death in some instances [18, 22]. The results of this study agree with those of Pradeep et al. [25] who reported significant reductions in shoot lengths and dry weights in an experiment to identify Mn-tolerant chickpea germplasms. Reduced shoot lengths as a result of Mn toxicity have also been reported in broad beans [43].

5.3 Screening for Acidity Tolerance Through Pot Experiments

Pot experiments were conducted to verify accession tolerance levels observed in hydroponic experiments. In screening for tolerance to Al toxicity, inconsistencies between results from the two experiments were observed. Based on RTI, accessions ICCV 11514, K036, ICCV 10515, and Saina1 were tolerant. 19 other accessions were moderately tolerant and only 4 sensitive. In contrast, no accession was tolerant at 15µM Al in hydroponic screening as 11 accessions including Saina 1 and ICCV 11514 were moderately tolerant and the rest sensitive. Based on RSL, ICCV 11514 and K036 were still tolerant, together with ICCV 00108 and ICCV 11316. No accessions were tolerant in hydroponic screening. The inconsistencies between pot and hydroponic experiments are attributed to the heterogeneous nature of soil that makes it difficult to provide precise Al treatments and hence the differing results. Additionally, ion interactions in soil may compound or alleviate Al toxicity challenges. In contrast, Baier et al. [44] found a strong correlation between hydroponic and soil experiments when screening wheat for tolerance to Al toxicity. Similarly, Nava et al. [45] concluded that results from hydroponic experiments and those from acid soils were similar when screening oat cultivars for tolerance to Al toxicity.

While inconsistencies between hydroponic and pot screening results were evident for most of the indices used, an exception was noted where pot RRL results correlated significantly (r = 0.8) with those from the hydroponic experiments at 15µM Al. Saina 1, ICCV 11504, ICCV 11514, K036, ICCV 11316, Chania 2, ICCV 07114, and ICCV 00108 emerged as the standout tolerant accessions in both experiments. This consistency is a result of RRL taking into account the mean root growth, unlike other indices such as RTI which was calculated from the root length at harvest and did not take into account root length variations at sowing. Therefore, RRL is a better indicator of accession tolerance to Al toxicity. In a similar study, Vance et al. (2021) determined RRL to be the most accurate discriminator of Al tolerance in chickpea screening.

In screening for tolerance to Mn toxicity, RSL measurements from the experiments correlated weakly and negatively, (r=-0.1) while RSW correlated weakly (r = 0.13). The weak correlation is attributed to differing levels of Mn ions present in the nutrient solution cultures and soil used in the experiments. Plants grown hydroponically were treated with 150 ppm while soil Mn ion content was 33.6 ppm. Other than pH, soil Mn content is also influenced by many factors including redox conditions and moisture levels [46]. Apart from soil properties, soil Mn availability is also controlled by plant characteristics and the interactions between roots and the rhizosphere [47].

In screening for tolerance to Al toxicity, accessions Saina 1, ICCV 11519, ICCV 11504, ICCV 11514, ICCV 11316, and ICCV 07114 showed promise. These accessions were either tolerant or moderately tolerant across all indices at 15µM Al. At 60 µM Al, Saina 1 based on RSL; ICCV 11316, ICCV 11514, and ICCV 07114, based on RRW; ICCV 11504 based on RSW; and ICCV 11519 based on RSL, RRW and RSW were moderately tolerant. The high tolerance levels observed in hydroponic experiments were supported by pot experiment results where Saina1 was tolerant based on RTI and RRL, ICCV 11316 based on RSL and RRL, ICCV 11504 and ICCV 07114 based on RRL, and ICCV 11514 based on RTI, RSL, and RRL. In screening for tolerance to Mn toxicity, accessions ICCV 07313, ICCV 07101, ICCV 97128, ICCV 11514, Chania 2, and Saina 1 were tolerant based RSL. Symptom scoring highlighted ICCV 11519, Saina 1, and ICCV 07313 as the standout accessions with the least Mn toxicity symptoms. Overall, accessions Saina 1 and ICCV 11514 stood out for tolerance to Al and Mn toxicity across all indices. These accessions emerged as the promising lines for promotion among farmers.

Funding

Part funding of this study was through the Australian Awards fellowship awarded to the second author for his postgraduate studies.

Conflicts of interest

The authors have no competing interests to declare that are relevant to the content of this article.

Ethics and consent to participate

Chickpea seed collection for this study was complied with the Seeds and Plant Varieties Act of the Kenyan Constitution.

Data availability

The authors declare that the data supporting the findings of this study are available within the paper. Should any raw data files be needed in another format they are available from the corresponding author upon reasonable request.

Code availability

Not applicable

Authors' contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by the first author and second author. The first draft of the manuscript was written by the first author and second authors and the third author commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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Assessment of Selected Chickpea Accessions for Tolerance to Aluminium and Manganese Toxicities in Solution Culture and Pot Experiments

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Abstract

Figures

Introduction

Materials and Methods

3.1 Plant Materials and germination tests

3.2 Experiment 1: Screening for tolerance to Al toxicity

3.2.1 Accession Sampling, Measurements, and Calculation of Indices

3.3 Experiment 2: Screening for tolerance to Mn toxicity

3.3.1 Accession Sampling, Measurements, and Symptom Scoring

3.4 Experiment 3: Screening for tolerance to soil acidity in pot experiments

3.4.1 Accession Sampling, Measurements, and Calculation of Indices

3.5 Plant Analysis

3.6 Statistical analysis

Results

4.1 Screening for Tolerance to Aluminium Toxicity

4.1.1 Relative Tolerance Index

4.1.2 Relative Change in Root Length

4.1.3 Relative Shoot Length

4.1.4 Relative Root Weight

4.1.5 Relative Shoot Weights

4.2 Screening for Tolerance to Manganese Toxicity

4.2.1 Symptom Scores

4.2.2 Relative shoot length (RSL) and Relative shoot weights (RSW)

4.3 Screening for Acidity Tolerance Through Pot Experiments

4.3.1 Relative Tolerance Index and Relative Root Length

4.3.2 Relative Shoot Length

4.3.3 Relative Root and Shoot Weights

Discussion

5.1 Screening for Tolerance to Aluminium Toxicity

5.2 Screening for Tolerance to Manganese Toxicity

5.3 Screening for Acidity Tolerance Through Pot Experiments

Conclusions and Recommendations

Declarations

Funding

Conflicts of interest

Ethics and consent to participate

Data availability

Code availability

Authors' contributions

References

Additional Declarations

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