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Article

Development and Metabolic Characterization of Horse Gram (Macrotyloma uniflorum Lam. (Verdc.)) Mutants for Powdery Mildew Resistance

by
Rajaprakasam Sudhagar
1,*,
Shanmugavel Priyanka
2,
Vanniarajan Chockalingam
3,
Vaithiyanathan Sendhilvel
4,
Jegadeesan Souframanien
5,
Kalimuthu Raja
6 and
Selvaraju Kanagarajan
7,*
1
Sugarcane Research Station, Tamil Nadu Agricultural University, Vellore 635806, Tamil Nadu, India
2
Centre for Plant Breeding and Genetics, Tamil Nadu Agricultural University, Coimbatore 641003, Tamil Nadu, India
3
Department of Plant Breeding and Genetics, Agricultural College & Research Institute, Agricultural University, Madurai 625104, Tamil Nadu, India
4
Department of Plant Pathology, Centre for Plant Protection Studies, Tamil Nadu Agricultural University, Coimbatore 641003, Tamil Nadu, India
5
Bhabha Atomic Research Centre, Government of India, Trombay, Mumbai 400085, Maharashtra, India
6
Department of Nanoscience and Technology, Tamil Nadu Agricultural University, Coimbatore 641003, Tamil Nadu, India
7
Department of Plant Breeding, Swedish University of Agricultural Sciences, P.O. Box 190, 234 22 Lomma, Sweden
*
Authors to whom correspondence should be addressed.
Agronomy 2022, 12(4), 800; https://doi.org/10.3390/agronomy12040800
Submission received: 2 February 2022 / Revised: 18 March 2022 / Accepted: 25 March 2022 / Published: 26 March 2022
Browse Figures
Versions Notes

Abstract

:
Horse gram is one of the lesser-known beans widely grown in India. One hundred and twenty-three homozygous horse gram mutants were screened for powdery mildew (PM) disease resistance using the grade 0 to 4. The mutants were grouped based on the disease level of 0 to 2 (resistant) and susceptible (3 to 4). The PM altered the chlorophyll fluorescence (a/b ratio), maturity duration, and yield attributing traits. The yield loss ranged from 4.55% to 72.66%. After affirming the resistance level, the resistant mutant (RM) with minimum yield loss (scale:0) and the susceptible mutant (SM) with maximum loss (scale:4) were used for metabolomic analysis through GC-MS. PM infection induced expression of 66 metabolites representing 32 functional classes. The number of unique classes in RM and SM was 13 and 11, respectively, while eight were common. A fold change in the common metabolites indicated an enhanced accumulation of amine, alcohol, and ester in RM. Along with pathogen-induced defensive metabolites, RM produced silane and fluorene, whose biological significance in disease resistance is unknown. Though SM expressed defence-related bio-molecules, it failed to yield better.

1. Introduction

Legumes are cultivated in two main seasons, Kharif (June–September) and Rabi (September/October–December) in southern India. Legumes, such as green gram, black gram, and red gram, are usually cultivated during the Kharif season. The Rabi season coincides with the winter season, and therefore dew-loving legumes, such as chickpea and horse gram, are commonly grown. Generally, the rainy days are few during the Rabi season, reinforcing the cultivation of medium-duration and less water-dependent crops such as arid legumes. Horse gram (Macrotyloma uniflorum (Lam.) Verdc.) is one such important diploid, cool-season arid legume cultivated in dry and marginal lands. This legume is grown as a contingent crop in accordance with the completion of the northeast monsoon (Rabi season) to utilize the residual moisture. This crop is preferred by the marginal farmers with poor land resources owing to its multi-utility [1]. Horse gram cultivation accomplishes many functions, such as (i) sustains nutritional sustainability in the arid region; (ii) breaks the cereal disease continuum; (iii) enriches the soil nitrogen through root nodules; (iv) enriches the soil organic carbon through leaf litter; (v) avoids soil erosion as a cover crop; and (vi) produce nutritious fodder. Horse gram can reclaim cultivable wastelands into a Nutri garden. The nutrient pre-eminence of horse gram includes significant protein, lysine, and micronutrient contents. Being a neglected crop, it provides many nutrients, as it is highly essential for humans. It prevents us from various types of dreadful diseases. The therapeutic potential of horse gram has been well retrieved from the ancient literature [2]. Strict self-pollination, narrow genetic variability for economic traits, and fewer studies on germplasm characterization urge accelerated breeding programs in horse gram as it has been identified to be a promising crop of the future [3]. The success of classical hybridization to create genetic variation in horse gram is limited due to its small flowers, closed floral structure, prostrating growth habit, and significant flower dropping. The potential of induced mutagenesis in the evolution of the variability in economic traits for horse gram has been reported [4]. Powdery mildew challenges photosynthesis and alters routine physiological processes, thereby significantly reducing horse gram yield [5].
Plants express stress-responsive transcripts and synthesize compatible metabolites to sustain stressed conditions [6]. Extensive transcriptomic studies were carried out to understand the role of different metabolic pathways under stress [7]. Metabolomics is an authoritative tool that assists in analysing the stress-induced metabolic changes that supplement the transcriptome data and thus helps develop or identify stress-tolerant genotypes. Metabolite profiling through the GC-MS technique is widely used to understand the metabolite changes under stress [8]. Stress-induced metabolic changes act as osmolytes, antioxidants, chelating agents, maintain cell membranes, iron transport, and turgidity, and also stabilize proteins and enzyme biosynthesis [9]. These up/down regulations of bio-molecules are the energy budgeting strategies evolved by plants to deal with the stress [10]. Reports on the resistance sources for E. polygoni in horse gram are rare.
Further, the knowledge on E. polygoni-induced metabolic changes is still unexplored. The tolerance mechanism for E. polygoni can be deciphered through comparative metabolomic studies between resistant and susceptible genotypes. Therefore, the present study aimed to classify the horse gram mutants into different powdery mildew resistant groups and comparative metabolomic profiling of resistance for further utilization.

2. Materials and Methods

2.1. Genetic Material and Categorization for Powdery Mildew Resistance

2.1.1. Evolution of Genetic Materials

Two mutant populations were developed by mutating the popular horse gram varieties PAIYUR 2 and CRIDA 1–18 R.
The mutagens were gamma rays (G) (100–400 Gy), electron beam (EB) (100–400 Gy), G + EB (100–400 Gy), and a combination of G and ethyl methanesulfonate (EMS) (G:100–400 Gy + EMS: 0.3%). In each treatment, 500 seeds were mutated. The M1 and M2 generations were sown during Rabi 2017 and 2018, respectively. The mutant populations were handled separately. All the M1 plants were harvested individually and forwarded to M2. The mutagenic efficiency and effectiveness were worked out based on the chlorophyll mutants and injuries/sterility. The promising M2 segregants were harvested individually and forwarded on a plant to row basis to M3 during Rabi 2019. The M1, M2, and M3 experiments were conducted at the Department of Pulses, Centre for Plant Breeding and Genetics, Tamil Nadu Agricultural University, Coimbatore, India.

2.1.2. Screening of Mutants for Powdery Mildew Resistance

Of the total mutants evolved, a total of 123 homozygous M3 promising mutants were alone considered for powdery mildew resistance screening under natural epiphytotic conditions (Table S1) during the Rabi (winter) season 2019. The climatic parameters are shown in Table S2.
The statistical design and replications were randomized block design (RBD) and three, respectively. All the recommended packages in current practices were followed. The conidia of E. polygoni were collected from the infected plants raised under greenhouse condition in sterile water using a soft brush. A suspension of conidia containing about 1 × 106 per mL was prepared separately in sterile water using a haemocytometer. The 25-day-old crop was inoculated by spraying this suspension with a hand sprayer at 4 PM for powdery mildew infection at vegetative growth. An equal number of plants were left un-inoculated to serve as a control. The plots were not sprayed with plant protection chemicals. The mutants were classified into varied resistant classes based on a 0–4 scale, as advocated by [11,12,13]. All plants in replication were considered for grading 17 days after inoculation. The description of the grading is given in Table 1.

2.1.3. Pathogen Confirmation and Classification of Mutants

The conidial confirmation and density observation were done using a phase-contrast microscope (PCM) and scanning electron microscopes (SEM). For the SEM analysis, the samples were coated with a low-vacuum sputter coating machine (EMITECH (SC7620) and mounted on a specimen stub. The FEI QUANTA 250 instrument was used for fruiting body identification. Five plants from each mutant were randomly tagged, and per plant yield was calculated. The percent yield loss was calculated by the formula: yield of inoculated mutant/yield of respective control × 100. This preliminary data was utilized to select extreme mutants for further testing under both fields and controlled conditions in the subsequent generation. The resistant (scale 0, 1 and 2) and susceptible (scale 3 and 4) group mutants were harvested separately.

2.1.4. Confirmation of Powdery Mildew Resistance and Tagging of Mutants for Metabolomic Analysis

In the subsequent year, the powdery mildew resistance of the tagged mutants was re-established by both confirmative field and shade-net trails during Rabi 2020. The experiments were conducted at the Sugarcane Research Station, Melalathur, Tamil Nadu Agricultural University. The weather parameters are shown in Table S2. For the confirmative field trial, a field grown with horse gram in the previous season and in situ ploughed with powdery mildew infected residues was selected. A total of three isolated resistant (scale:0) and nine susceptible (scale:4) mutants were sown in three replications in RBD. Each entry was sown in five rows. The susceptible mutant with maximum yield loss in the previous year’s trial (TNAU-HG-M004) was sown in two rows after every resistant entry to ensure adequate inoculum load. To affirm disease prevalence at the appropriate crop growth stage, TNAU-HG-M004 was sown twenty days earlier than the resistant mutants. The same set of entries without inter-planting of TNAU-HG-004 was sown in a control plot. The conidium collected from TNAU-HG-M004 was tapped over other mutants at the vegetative stage. During the study period, no plant protection chemicals were sprayed. To estimate the powdery mildew induced economic loss, the yield and its attributing traits were recorded in five randomly selected plants in all replications. The same set of mutants was sown in pots as a second confirmative trial in a completely randomized block design in a shade-net study. The soil from the inoculum-loaded experimental field was utilized to prepare the potting mixture. Each entry was replicated thrice, and five pots were sown per replication. At the vegetative stage, powdery mildew-infected leaves of TNAU-HG-004 were stapled on ten randomly selected leaves of other mutants. The scaling technique for both the field and pot studies was adopted as described in Table 1. Five plants from each mutant were randomly tagged, and the powdery mildew induced changes in chlorophyll fluorescence (a/b ratio), crop maturity duration, and yield were documented. The chlorophyll fluorescence (a/b ratio) was estimated using an OS1p+ chlorophyll fluorometer (Optic sciences). At harvest, the resistant mutant with the minimum yield loss and the susceptible mutant with the maximum yield loss were tagged, compared, and utilized for metabolomic studies through GC-MS.

2.1.5. Data Analysis

The data pertinent to grouping and tagging of extreme mutants were analysed using SPSS version 12 for estimating the standard error of the mean.

2.2. Metabolite Extraction and GC-MS Analysis

Leaf samples from the pot-grown resistant mutant with minimum powdery mildew-induced yield loss (TNAU-HG-M11) and the susceptible mutant with maximum yield loss (TNAU-HG-M4) were collected after 17 days of inoculation for metabolomic analyses. Leaf tissues in three replications immediately after collection were flash-frozen in liquid nitrogen. Fresh frozen leaf tissues (40 mg) were ground using liquid nitrogen, extracted with methanol followed by chloroform [14]. An aliquot of 50 µL ribitol (0.2 mg/mL in water) was utilized as internal standard and filtered through a PVDF syringe filter (0.22 µm) (Millipore, Dublin, Ireland). Speed Vac was used to concentrate the extracts. N-methyl-N-[trimethylsilyl] trifluoroacetoamide (80 μL; MSTFA) was used for trimethylsilylation. The temperature and time of trimethylsilylation were 37 °C and 30 min, respectively. The samples (about 1 µL) were injected into a GC injection port (AI3000 II, Thermo Fischer Scientific, Waltham, MA, USA) connected to a GC-MS (TRACE™ GC Ultra with DSQII Quadrupole mass spectrometer). The technical specifications of the capillary column are 30 cm length, 0.25 mm diameter, and 0.25 µm film thickness-Agilent, DB-5MS Ultra Inert, and 122–5532. The injection port temperature was maintained at 250 °C. The oven temperature was programmed at 70 °C (5 min hold) and increased 5 °C per minute to 300 °C. Helium was the carrier gas, and the flow rate was 1 mL/s. The temperature at the electron ionization source was maintained at 250 °C. The abundance of ions in the range of 50–600 m/z was scanned at a rate of 1.1 scans per second. The NIST library was used to identify the compounds, and the data were analysed using R software.

3. Results and Discussion

3.1. Powdery Mildew-Induced Changes in the Yield Attributing Traits

A total of 123 homozygous horse gram mutants (Table S1) were screened for powdery mildew resistance under field conditions during the Rabi season 2019. The mutants were free from powdery mildew infection up to 35 days after sowing. The infection started when a shading effect formed between leaves in the cool dew days. The climatic conditions are provided in Table S2. Initially, small, white, powdery spots appeared on the upper surface of the leaves, and the size of the spots progressed along with the age of the crop. Later, the infection spreads onto the lower leaf surface; the leaves become brown, brittle, the petiole becomes weak, broken at the joints, and the leaves are forced into premature senescence (Figure S1a–f). During the third stage of infection, a white powdery mass developed on the pods (Figure S2a,b). A visible difference was noticed between the resistant and susceptible genotypes (Figure 1A,B). From the susceptible mutant, white powdery masses were collected for species identification. Based on the morphology of the conidiophores and conidia of the pathogen, it was identified as E. polygoni (Figure 1C,D). It was observed that barrel-shaped conidia were borne on the conidiophores in chains. The conidiophores were short, initially cylindrical, and later changed to barrel-shaped, and became elliptical at disposal.
Based on the level of infection, the mutants were grouped into four categories: Scale 0, Scale 1, Scale 2, Scale 3, and Scale 4. It was evident that the higher the degrees of infection, the fewer seeds per pod. The number of resistant mutants with the Scale 0, Scale 1, and Scale 2 levels of infection were three, four, and eleven, respectively. The susceptible mutants with a Scale 3 and Scale 4 grade of infection were ninety-six and nine (Figure 2A), respectively.
During the first-year field trial, the mean data on E. polygoni-induced single plant yield loss was assessed (Table S3) to identify the distinctive extreme mutants in the Scale 0 and 4 categories. The range of yield loss was 4.98 to 5.28% (Scale 0); 7.62 to 9.94% (Scale 1); 10.99 to 20.14% (Scale 2); 21.0 to 41.0% (Scale 3); and 42.88 to 71.86% (Scale 4). The mutants of Scale 0 and Scale 4 were utilized for the second-year confirmative trials. The climatic conditions are shown in Table S2. To assess the host physiology of E. polygoni infection in horse gram, the data on chlorophyll fluorescence (a/b ratio), number of flowering clusters per plant, number of pods per plant, number of seeds per pod, days to maturity, and single plant were recorded both under field and shade-net conditions. The results of second-year screening experiments confirmed the findings of the first-year trial. In the Scale 0 group, the mutant TNAU-HG-M11 (RM) had minimum yield loss, and TNAU-HG-M4 (SM) exhibited the maximum yield loss in the Scale 4 category (Figure 2B,C).
No significant variation was noticed in the character expression of RM and SM between the field and shade-net experiments. The RM performed well in the field and shade-net experiments, and vice versa indicated the genetic influence (Figures S3 and S4). The net house studies and their data are utilized to discuss the result precisely, and the same was compared with field data for drawing meaningful inferences (Table 2).
The leaves of RM were dark-green under the inoculated condition, both in the field and shade-net experiments; on the contrary, SM had dull-green leaves. This abridged chlorophyll concentration resulted in a significant variation in chlorophyll fluorescence (a/b ratio) of SM. It was 0.33 and 0.77 under inoculated and controlled conditions, respectively, while no considerable variation was noticed in RM. The ability of E. polygoni to reduce the photosynthetic rate in horse gram has been reported [5]. E. polygoni infection resulted in a forced maturity in SM. It matured in 117.8 days in the un-inoculated condition, but it matured in 72.40 days under infested conditions. The RM maintained the same crop duration in both situations. The reduced chlorophyll fluorescence and crop duration in SM resulted in a significant variation in yield attributing traits. It had an average number of 56.20 flower-bearing clusters per plant, which led to a more substantial decrease in the associated number of pods (61.20) and number of seeds per pod (1.20). It ultimately reduced the single plant yield (14.60 g). Nevertheless, RM maintained its yield level. Therefore, these two extreme mutants were utilized to understand the powdery mildew-induced metabolic changes in horse gram.

3.2. Metabolomic Analysis through GC-MS Analysis

Many diseases infect plants, and thereby, significant economic loss is witnessed. Pathogen infection triggers immune response(s) in plants due to expression/activation of various metabolites. Plants with activated immune responses could survive and yield in the pathogen-challenged environments; otherwise, they succumb to the disease. In the present study, RM and SM were utilized to understand the bio-molecule production pattern in response to E. polygoni infection through solvent extraction followed by GC-MS. The GC-MS produced typical chromatograms between RM and SM (Figure S5a,b).
The mutants expressed both exclusive and common metabolites (Figure 3).
A total of 66 bio-molecules representing 32 functional classes were expressed. The RM and SM expressed 29 and 32 exclusive metabolites, respectively. Of the five common metabolites, three were upregulated.
The details of bio-molecules and functional classes are given in Table 3 and Table S4, respectively.
The RM exhibited exclusive expression of thirteen metabolite classes while the SM expressed eleven. A total of 10 common metabolite classes, namely, alcohol, alkane, amide, amine, carboxylic acid, ester, ketone, and sugar, were expressed in SM and RM. A varied quantum of expression was noticed between the categories. The goodness of fit of the bio-molecules with the NIST library is shown in Table S5.
A total of seven good and two fair matches were identified in the SM, while seven good and five fair matches were observed in the RM. The bio-molecules decane, propanoic acid, n-Hexadecanoic acid, polygalitol, and 3-o-Methyl-d-Glucose were commonly expressed in both SM and RM, and the fold change is presented in Figure 4.
The fold change was not significant, except for 3-o-Methyl-d-Glucose (sugar) and polygalitol (alcohol). The expression of the former was significant in SM while the latter was in RM.

3.3. Biological Significance of Unique Metabolites Expressed in the Resistant Mutant

In RM, a total of 13 exclusive classes of compounds were expressed (Figure 5A). The order of expression was siloxane (3.91%), furan (1.58%), benzoic acid (0.86%), and aromatic dicarboxylic acid (0.60%). The other resistance-specific, narrow-ranged metabolites were organochloride (0.38%), oxine (0.36%), nitrile (0.34%), piperidine (0.30%), acetate (0.29%), fluorene (0.28%), aldehyde (0.27%), and benzodiazepines (0.27%). These bio-molecules, either individually and/or in combination, helped the RM to resist the pathogen development, spread, and therefore finally yielded better at harvest (Table 2).
Siloxane was the most significantly expressed bio-molecule in the RM. Siloxane or silicon (Si) are previously considered as non-essential for plant growth and development [15]; however, application of Si improved plant vigour and resistance against diseases [16]. Si imparts disease resistance through the formation of physical barriers [16], signal transduction [17], activation of defence compounds [18], and enzymes [19]. The anti-microbial properties of furan derivatives were earlier reported by [20]. The furan derivatives are synthesized in leaves constantly and exhibit strong free radical scavenging capacity [21]. Therefore, it is inferred that RM counteracted the E. polygoni-induced oxidative stress in the leaves through a high accumulation of furan.
Plants produce a variety of benzoic acid (BA) compounds, including aromatic dicarboxylic acid, to sustain various stresses. The BAs involved in signal transduction induced systemic acquired resistance [22], regulate the stress-induced forced senescence [23], and promote and shoot growth [24] in challenging environments. Expression of organochlorine was observed in the RM. Natural halogenated compounds were isolated commonly from marine ecosystem-borne microorganisms [25]. However, very few organochlorine compounds were isolated from higher plants [26]. The synthesis of organochlorine was reported under the diseased condition in lily with antifungal activity [27].
Oximes (R1R2C=NOH) are the volatile compounds produced in response to the environment [28], and act as herbivore deterrents [29], are attractants for natural enemies [30], and are ATP production inhibitors in microorganisms [31,32]. Oximes are converted to the corresponding alcohols, aldehydes, carboxylic acids, and glucosides, thereby conferring resistance to plants. Further, oximes regulate the production of plant hormones IAA and indole-3-acetaldoxime [33,34,35] and thus maintain plant growth under stress. Oxime expression was witnessed only in RM; therefore, one can hypothesize that it might have (i) helped in the regulation of auxin synthesis in RM, as it maintained good crop growth under powdery mildew infection (Table 2); and (ii) inhibited ATP production in E. polygoni as it failed to survive (Figures S3–S5), and part of the oximes might have been converted and stored as aldehyde. Nevertheless, aldehyde expression was not noticed in SM (Figure 4). Aldehyde-induced expression of plant defence genes was reported by [36]. On the contrary, Hansjakob [37] reported that aldehyde helped in powdery mildew spore germination.
Oximes are present in E and Z forms; the former synthesizes cyanogenic glucoside and later glucosinolate. Certain microorganisms convert Z oximes into corresponding nitriles, thereby escaping from the toxic effects of oximes [38], and therefore readily infect the plant. In the present study, E. polygoni converted a part of the oxime into nitrile (Table 3) in RM, but the mutant performed well with the help of other defensive metabolites. Piperidine is toxic and has antifeedant properties [39,40]. It is synthesised against herbivores in Lobelia cardinalis [41] and Conium maculatum [42]. However, in the present study, the expression of piperidine was reported against E. polygoni. The anti-microbial activity of acetic acid is well recognized as it influences the proton release in the membrane [43]. Expression of two unique metabolites, ‘silane’ and ‘fluorene’, were observed in RM. The biological significance of these bio-molecules in disease resistance is to be investigated. However, Lu et al. [44] reported the beneficial effect of silane in improving plant fibre quality and stability, and Roessingh et al. [45] observed the feeding and oviposition stimulant ability of fluorene.

3.4. Biological Significance of Unique Metabolites Expressed in the Susceptible Mutant

In SM, eleven unique metabolites were expressed (Figure 5B). The linear order of expression was silyl ether (2.04%), hydroquinone (1.67%), carotenoid (0.95%), alkene (0.90%), phenol (0.68%), nitrosourea (0.66%), aliphatic nitrile (0.61%), ether (0.5%), glucosinolate (0.35%), nitrogen compound (0.35%), and terpenoid (0.32%).
Silyl ether was the most predominant bio-molecule. Silyl ether compounds have a silicon (si) atom. Si mediates resistance to pathogen infection through signalling and defence gene expression [17], structural modifications [46], systemic acquired resistance, and production of antimicrobial compounds [18]. Si accumulates in epidermal tissue, forms complex organic compounds, and induces the production of phenolic compounds [47]. Expression of phenol was also noticed in the present quest.
Hydroquinones are a category of phenolic lipids that have salicylic acid (SA) as their phenolic group. The role of SA in disease resistance is well documented [48]. Hydroquinone inhibits disease development and improves survival and seedling vigour [49]. Terpenoids are the largest group of volatiles produced by the plants in response to insect and pathogen-induced stresses [50]. Mainly, terpenoids are produced under insect attack [51]. However, Prisic et al. [52] reported the role of terpenes as phytoalexins and their accumulation in response to pathogen infection. Carotenoids are the C40 terpenoids that play a significant role in photosynthesis, photo-protection, photomorphogenesis [53], signalling under oxidative stress [54], root development, and branching [55]. It is inferred that the SM tried to maintain the normal photosynthetic process under PM challenge through the expression of carotenoid.
The SM expressed a significant amount of alkene. The plant cuticle is made up of the long-chain fatty acid derivatives alkanes, alkenes, and secondary alcohols [56]. It provides resistance to pest and disease attacks and is associated with a few other plant developmental processes [57]. The role of alkene in disease resistance was reported by [58]. Expression of glucosinolate was observed in SM, which is a proven toxic metabolite against microorganisms and is widely reported in Brassicales [59]. A higher expression of aliphatic nitrile than glucosinolate was also noticed, indicating the ability of E. polygoni to detoxify glucosinolate. Such detoxification forced SM to succumb to E. polygoni. Two bio-molecules, 2-phenyl-1,3-dioxan-5-yl ester (an ether molecule) and nitrosourea, were expressed only in the SM, whose role in plant defence is to be investigated. However, the anti-oxidant ability of 2-phenyl-1,3-dioxan-5-yl ester was reported in mammals [60], and nitrosourea is a well-proven alkylating agent [61], mainly used as an anti-cancer drug.

3.5. Biological Significance of Expression of the Common Class of Bio-Molecules Expressed Both in Resistant and Susceptible Mutants

A total of eight classes of compounds, namely, alkane, alcohol, sugar, carboxylic acid, ketone, amine, amide, and ester, were commonly expressed in RM and SM (Figure 5C).
Sugar was the most expressed metabolite, indicating its vital role in stress alleviation. Earlier, the multi-functionality of sugars in signalling, metabolic gene activation, and immunity trigger was reported by [62,63]. Further, a common expression of carboxylic acid between RM and SM made liable to affirm the earlier findings of [64] as carboxylic acid regulates the activation of the sugar signalling pathways and, thus, confers resistance to biotic stress. Amine (18.7%) and alcohol (4.18%) were highly expressed in RM. Polyamines play a vital role in plant developmental processes from root growth to seed development [65]. The functions of amines under abiotic stress [66] and biotic stress [67] are well documented. The role of various amides in plant disease resistance has also been reported previously [68].
Epidermal cuticles impart resistance to biotic and abiotic stresses. The cuticle is formed by cutin and cuticular wax. Samuels et al. [69] reported stress-induced cuticular wax modification, the subsequent production of alcohols, alkanes, esters, aldehydes, ketones, and secondary metabolites, and their protective roles. In horse gram, E. polygoni induced the expression of the above-listed compounds, but the quantity varied between PM and SM. Despite the expression of eight common classes of compounds, whose role in plant–microorganism interaction is well documented, the SM failed to perform well under powdery mildew infection.

4. Conclusions

In vivo and net house studies on powdery mildew resistance screening in horse gram yielded two extreme classes of resistant and susceptible mutants. E. polygoni affected the expression of yield attributing traits. The RM yielded well despite the initial infection. However, the characterized powdery mildew symptoms were noticed in leaves and pods of SM, and, therefore, it yielded very low. Comparative GC-MS analyses revealed that the RM expressed thirteen classes of unique metabolites through which it resisted pathogenicity. Although E. polygoni infection upregulated unique and common stress-responsive metabolites in the SM, it failed to combat the pathogenicity. Stress-triggered immune responsive functions in the SM may not be fully functional, and/or a part of the metabolites might have been diverted for other plant metabolic processes to survive in the stressed situation, which was evidenced through the presence of a few poorly filled pods and newer flowers till harvest.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy12040800/s1, Figure S1: Stages of E. polygoni infection in horse gram leaves; Figure S2: Stages of E. polygoni infection in horse gram pods (a) Maturing pods (b) Impact of infection on seed yield; Figure S3: Powdery mildew resistance as field view. (a) powdery mildew susceptible and (b) resistant horse gram mutant; Figure S4: Performance of horse gram mutants under shade net conditions. (a) powdery mildew susceptible mutant and (b) powdery mildew resistant mutant; Figure S5: Chromatograms of (a) acetone extract of powdery mildew resistant horse gram mutant and (b) acetone extract of powdery mildew susceptible horse gram mutant; Table S1: Details of horse gram mutants screened for powdery mildew resistance; Table S2: The details of climatic conditions prevailed during powdery mildew resistance screening; Table S3: The percentage of yield loss due to E. polygoni infection in different resistant classes of horse gram (values are in mean + SEM); Table S4: Details of bio-molecules expressed in horse gram in response to powdery mildew infestation; Table S5: The goodness of fit of the extracted bio-molecules from powdery mildew resistant and susceptible genotypes based on SI and RSI values.

Author Contributions

Conceptualization, R.S., V.S. and J.S.; methodology, R.S., V.C., V.S., K.R. and S.K.; formal analysis, R.S., S.P., K.R. and V.S.; investigation, R.S. and S.P.; data curation, R.S., V.C, J.S. and V.S.; writing—original draft preparation, R.S., writing—review and editing, R.S., V.C., V.S., J.S. and S.K.; and supervision, V.C. and J.S. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding authors.

Acknowledgments

The authors are grateful to (i) S. Dutta, BARC—Government of India, for his technical guidance; (ii) M. Raveendran, Department of Plant Molecular Biology and Biotechnology, Tamil Nadu Agricultural University (TNAU), India, for his relentless scientific support; (iii) P. Jayamani, Department of Pulses, Centre for Plant Breeding and Genetics, TNAU, India, for her critical suggestions and scientific support; (iv) P. Jayakumar, TNAU, India, for his constant technical support; and (v) M. Senthil, Kandaswami Kandar’s College, ParamathiVelur, Tamil Nadu, India, for his technical expertise in the bio-molecule grouping.

Conflicts of Interest

The authors declare no conflict of interest.

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Agronomy 12 00800 g001 550
Figure 1. (A) Powdery mildew-resistant mutant; (B) powdery mildew-susceptible mutant; (C) Identification of the powdery mildew species: phase-contrast microscopy; (D) Identification of powdery mildew species: SEM.
Figure 1. (A) Powdery mildew-resistant mutant; (B) powdery mildew-susceptible mutant; (C) Identification of the powdery mildew species: phase-contrast microscopy; (D) Identification of powdery mildew species: SEM.
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Agronomy 12 00800 g002 550
Figure 2. (A) Categorization of horse gram mutants for powdery mildew resistance; (B) Percentage of yield loss in the resistant mutants (Scale 4) due to E. polygoni infection; (C) Percentage of yield loss in the susceptible mutants (Scale 4) due to E. polygoni infection.
Figure 2. (A) Categorization of horse gram mutants for powdery mildew resistance; (B) Percentage of yield loss in the resistant mutants (Scale 4) due to E. polygoni infection; (C) Percentage of yield loss in the susceptible mutants (Scale 4) due to E. polygoni infection.
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Agronomy 12 00800 g003 550
Figure 3. Number of metabolites accumulated in resistant and susceptible genotypes. Numbers in the parenthesis indicate upregulated (red) and downregulated metabolites (black).
Figure 3. Number of metabolites accumulated in resistant and susceptible genotypes. Numbers in the parenthesis indicate upregulated (red) and downregulated metabolites (black).
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Agronomy 12 00800 g004 550
Figure 4. Fold change analysis of the common metabolites.
Figure 4. Fold change analysis of the common metabolites.
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Figure 5. (A) Enhanced accumulation pattern of metabolites in the powdery mildew-resistant mutant; (B) Enhanced accumulation pattern of metabolites in the powdery mildew-susceptible mutant; (C) Accumulation pattern of common metabolites in the powdery mildew-resistant and -susceptible mutants.
Figure 5. (A) Enhanced accumulation pattern of metabolites in the powdery mildew-resistant mutant; (B) Enhanced accumulation pattern of metabolites in the powdery mildew-susceptible mutant; (C) Accumulation pattern of common metabolites in the powdery mildew-resistant and -susceptible mutants.
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Table
Table 1. Details of the scaling technique adopted for powdery mildew resistance screening in horse gram.
Table 1. Details of the scaling technique adopted for powdery mildew resistance screening in horse gram.
DescriptionScoreDisease Reaction
No symptoms of powdery mildew growth0R
Small and sparse mycelial growth. Visualization of non-significant sporulation1R
Macroscopic: Slight mycelia growth
Microscopic: Slight to moderate mycelia growth with conidiophores		2R
Macroscopic: Moderate mycelial growth
Microscopic: Moderate mycelial development with moderate to heavy sporulation		3S
Macroscopic: Heavy mycelia growth
Microscopic: Heavy mycelia growth with copious sporulation		4S
Table
Table 2. Effect of E. polygoni on the growth and yield attributing traits in horse gram (values are the mean ± SEM).
Table 2. Effect of E. polygoni on the growth and yield attributing traits in horse gram (values are the mean ± SEM).
TraitsFiled ConditionShade-Net Condition
InoculatedControlInoculatedControl
SMRMSMRMSMRMSMRM
1DuGDaGDaGDaGDuGDaGDaGDaG
20.32 ± 0.010.88 ± 0.010.86 ± 0.010.89 ± 0.010.33 ± 0.020.79 ± 0.020.77 ± 0.010.79 ± 0.01
373.80 ± 0.6678.20 ± 1.0474.40 ± 0.6182.00 ± 0.6356.20 ± 0.9672.80 ± 0.5274.80 ± 0.3479.60 ± 0.46
444.00 ± 0.75144.40 ± 1.00135.20 ± 0.52153.40 ± 0.6761.20 ± 0.77132.60 ± 0.46130.40 ± 0.47141.60 ± 0.61
51.40 ± 0.225.60 ± 0.225.20 ± 0.185.40 ± 0.221.20 ± 0.185.00 ± 0.284.80 ± 0.345.60 ± 0.22
674.00 ± 1.10119.00 ± 0.63121.00 ± 0.52124.40 ± 0.6172.40 ± 0.73116.60 ± 0.46117.80 ± 0.52119.00 ± 0.63
714.12 ± 0.7350.40 ± 0.9252.20 ± 0.7752.80 ± 0.7714.60 ± 0.4646.80 ± 0.5950.20 ± 0.7750.60 ± 0.61
SM—Susceptible mutant. RM—Resistant mutant. DuG—Dull green. DaG—Dark green. 1—Leaf colour. 2—Chlorophyll fluorescence (a/b ratio). 3—No. flowering clusters/plant. 4—No. of pods/plant. 5—No. of seeds/pod. 6—Days to maturity. 7—Single plant yield (g).
Table
Table 3. List of differentially expressed bio-molecules classes in horse gram with respect to powdery mildew infection.
Table 3. List of differentially expressed bio-molecules classes in horse gram with respect to powdery mildew infection.
S. NoClassPeak Area (%)S. NoClassPeak Area (%)
RSRS
1Acetate0.29-17Furan1.58-
2Alcohol4.180.3018Glucosinolate-0.35
3Aldehyde0.27-19Hydroquinone-1.67
4Aliphatic nitrile-0.6120Ketone0.790.86
5Alkane0.491.4221Nitrile0.34-
6Alkene-0.9022Nitrogen compound-0.35
7Amide0.430.5623Nitrosourea-0.66
8Amine18.708.4624Organochloride0.38-
9Aromatic dicarboxylic acid0.60-25Oxime0.36-
10Benzodiazepines0.27-26Phenol-0.68
11Benzoic acid0.86-27Piperidine0.30-
12Carboxylic acid5.306.528Silane0.30-
13Carotenoid-0.9529Siloxane3.91-
14Ester0.370.2630Silyl ether-2.04
15Ether-0.5031Sugar18.4722.79
16Fluorene0.28-32Terpenoid-0.32
S—Susceptible. R—Resistant.
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Sudhagar, R.; Priyanka, S.; Chockalingam, V.; Sendhilvel, V.; Souframanien, J.; Raja, K.; Kanagarajan, S. Development and Metabolic Characterization of Horse Gram (Macrotyloma uniflorum Lam. (Verdc.)) Mutants for Powdery Mildew Resistance. Agronomy 2022, 12, 800. https://doi.org/10.3390/agronomy12040800

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Sudhagar R, Priyanka S, Chockalingam V, Sendhilvel V, Souframanien J, Raja K, Kanagarajan S. Development and Metabolic Characterization of Horse Gram (Macrotyloma uniflorum Lam. (Verdc.)) Mutants for Powdery Mildew Resistance. Agronomy. 2022; 12(4):800. https://doi.org/10.3390/agronomy12040800

Chicago/Turabian Style

Sudhagar, Rajaprakasam, Shanmugavel Priyanka, Vanniarajan Chockalingam, Vaithiyanathan Sendhilvel, Jegadeesan Souframanien, Kalimuthu Raja, and Selvaraju Kanagarajan. 2022. "Development and Metabolic Characterization of Horse Gram (Macrotyloma uniflorum Lam. (Verdc.)) Mutants for Powdery Mildew Resistance" Agronomy 12, no. 4: 800. https://doi.org/10.3390/agronomy12040800

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