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A study on preventive effects of Lawsonia inermis L. bioformulations against leaf spot

disease of maize

Tansukh Barupal, Mukesh Meena, Kanika Sharma

PII: S1878-8181(19)30940-5
DOI: https://doi.org/10.1016/j.bcab.2019.101473
Reference: BCAB 101473

To appear in: Biocatalysis and Agricultural Biotechnology

Received Date: 1 July 2019

Revised Date: 3 December 2019
Accepted Date: 13 December 2019

Please cite this article as: Barupal, T., Meena, M., Sharma, K., A study on preventive effects of
Lawsonia inermis L. bioformulations against leaf spot disease of maize, Biocatalysis and Agricultural
Biotechnology (2020), doi: https://doi.org/10.1016/j.bcab.2019.101473.

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1 A study on preventive effects of Lawsonia inermis L. bioformulations against leaf spot
2 disease of maize
3 Tansukh Barupal, Mukesh Meena* and Kanika Sharma
4 Department of Botany, Mohanlal Sukhadia University, Udaipur-313001, Rajasthan, India
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23 Corresponding author:
24 Dr. Mukesh Meena*
25 Department of Botany,
26 Mohanlal Sukhadia University,
27 Udaipur – 313001, Rajasthan, India
28 Email: mukeshmeenamlsu@gmail.com; drmukeshmeena321@mlsu.ac.in

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30 Abstract
31 Leaf spot disease caused by Curvularia lunata is one of the major constraints affecting the
32 cultivation of maize in India. Recently, it has been reported that the severity of curvularia leaf
33 spot was prevalent in moderate to severe intensities and cause extensive damage to the crop thus
34 lowering the yields. In the present study, in vivo preventive effect of Lawsonia inermis Linn.
35 bioformulation on curvularia leaf spot disease of maize has been studied. All the experiments
36 were conducted in pots. Percent disease index (PDI), percent disease control (PEDC) and others
37 various growth characteristics like plant height, number of leaves per plant, total carbohydrate
38 and protein content were recorded. Percent seed germination was also observed for seeds treated
39 with all formulations. A significant control of leaf spot disease was recorded with
40 bioformulations treatment T3 [seeds were treated with partially purified acetone extract (4 ml):
41 100% clove bud oil cake (4 ml): 100% cow dung (2 ml)], T4 [seeds were treated with partially
42 purified acetone extract (3 ml): 100% clove bud oil cake (4 ml): 100% cow dung (3 ml)], T2
43 [seeds were treated with 100% alcoholic crude extract (2ml): 100% clove bud oil cake (6 ml):
44 100% cow dung (2 ml)] and T1 [seeds were treated with 100% alcoholic crude extract (4 ml):
45 100% clove bud oil cake (4 ml): 100% cow dung (2 ml)] as compared to other bioformulation
46 treatments and its PDI and PEDC were recorded 9.10%, 10.00%, 17.50%, 19.00% and 89.47%,
47 88.43%, 79.76%, 78.03%, respectively. Results showed that treatment with bioformulation
48 treatment T3, notably increased plant height, number of leaves per plant, chlorophyll,
49 carotenoids, and total soluble sugar content followed by formulations number T4, T2, and T1.
50 However, total soluble protein content was observed to be less in T3 as compared to control.
51 This study suggests that these bioformulations could be essential towards sustainable agricultural
52 science deprived of harming the ecosystem. The synthesized bioformulations have enormous
53 potential to be commercially explored for agriculture use.
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55 Keywords: Lawsonia inermis; Maize; Bioformulations; Curvularia lunata; Disease; Mancozeb
56
57 Abbreviations: MMT, Million metric tons; PDA, Potato dextrose agar; BOD, Bio-oxygen
58 demand; SBM, Standard blotter method; PDI, Percent disease index; PEDC, Percent efficiency

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59 of disease control; SE, Standard error; CD, Critical difference; CV, Coefficient of variation;
60 CBB, Coomassie brilliant blue
61
62 Introduction
63 Maize (Zea mays L.) is the maximum resourceful crop with more extensive consistence in
64 diverse agro-ecologies. Around the world, it is cultivated on nearly 150 m ha in around 160
65 nations having more extensive assorted variety of environment, soil, biodiversity and managing
66 rehearses that contribute to 36% in the overall cereal production. United States of America,
67 Argentina, Brazil, China, Mexico, and India are the large-scale maize growing countries in the
68 globe. Amongst these nations, United States of America (USA) is the major producer of maize in
69 the world and exports 20% of its annual production. The average production in USA, China,
70 Brazil, India, Argentina, Ukraine, Mexico, Indonesia, France, and South Africa is 377.5, 224.9,
71 83.0, 44.3, 40.0, 39.2, 32.6, 19.0, 17.1 and 15.5 million metric tons (MMT), respectively
72 (Oishimaya, 2017; https://www.worldatlas.com/articles/world-leaders-in-corn-maize-production-
73 by-country.html).
74 In India, maize is the third furthermost vital nourishment crop next to rice and wheat. It is
75 cultivated throughout the year in different parts of the country for various purposes such as grain,
76 green cobs, fodder, baby-corn, sweet-corn, pop-corn and industrial purposes. The main maize
77 producing states are Karnataka, Madhya Pradesh, Bihar, Tamilnadu, Telangana, Maharashtra,
78 Andhra Pradesh, Uttar Pradesh, and Rajasthan (Agriculture-Statistical Year Book India,
79 mospi.nic.in/statistical-year-book-india/2017/177).
80 Maize is susceptible to various fungal infections (Barupal and Sharma, 2015). Among
81 these, curvularia leaf spot is frequent, extensive, damaging and an economically vital disease (Li
82 et al., 2003; Barupal et al., 2019). The disease caused broad destruction to the crop by affecting
83 the yields. The disease severity extended from 20.50 to 63.60 percent. This disease is caused by a
84 soil and a seed borne fungus Curvularia lunata (Wakker) Boedjn (Bisht et al., 2013). The
85 symptoms of the disease are the presence of small (1-6 mm) chlorotic lesions leading to necrotic
86 spots with grey halo (Dai et al., 1995; Akinbode, 2010). It is categorized by the development of
87 brown geniculate conidiophores with curved (arched) conidia in host tissue and culture media.
88 Scientists have been used both biological and chemical methods to preclude destruction
89 caused by C. lunata in the maize. The chemical method is widely used because of its diverse use

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 90 and ease of synthesis. Johnston et al. (1957) suggested that trichloromethyl arenethio sulphonates
91 control leaf spot of maize. It has been reported that carbendazim (bavistin) is effective for
92 diseases caused Fusarium spp. and Botrytis spp. which were found associated with fruit rot
93 diseases (Mishra 1988; Raju and Rao, 1989; Azad et al., 1992; Al-Said et al., 2011; Meena and
94 Zehra, 2019). The growth of the pathogen was completely inhibited by propiconazole (tilt) and
95 copper oxychloride (blitox) at 250 µg/ml and 500 µg/ml concentrations. But, now it has been
96 comprehended that chemically synthesized fungicides are affected to non-target organisms and
97 also cause severe environmental complications (Holmes and Eckert, 1999; Makarov et al., 2014;
98 El Gendy et al., 2015; Ranga Rao et al., 2015; Saharan et al., 2015). The toxicity of fungicide
99 azoxystrobin has been reported against invertebrates, fish and fresh water algae (Pest
100 Management Regulatory Agency, 2007; Ochoa-Acuna et al., 2009). There are several effects of
101 different chemical fungicides on mammals include cancer (propiconazole and triadimefon),
102 altered hepatic enzymes (propiconazole, myclobutanil and triadimefon) and altered reproduction
103 (myclobutanil and triadimefon) (Chen et al., 2009; Goetz and Dix, 2009; Meena et al., 2019a, b).
104 Therefore, at the present time our attention is moving in the direction of biological
105 methods to regulate plant diseases as they have no antagonistic consequence on humans and
106 environment. Adeleye and Ikotun (1989) investigated that extracts of a wild variety of Dioscorea
107 bulbifera L. have particular antifungal activity against C. lunata. Ulaganathan and Basha (2002)
108 reported that a soil bacterium, Bacillus sp. strain BC121 revealed immense antagonistic activity
109 against the C. lunata. Use of biological fungicides in control of plant diseases is gaining
110 importance day by day because they are cost-effective and eco-friendly (Golden, 2001; Pal and
111 Kumar, 2013; Meena et al., 2018a, b).
112 Currently, plant-based antifungal formulations are used to manage the diseases of the
113 plants. These antifungal formulations comprise effective compounds accompanied by specific
114 inert carrier material (Omer, 2010). Anis et al. (2012) established a Na – alginate based
115 bioformulation to control the charcoal rot disease of sunflower. It has been evaluated the
116 efficiency of plant extracts formulations to control Fusarium oxysporium caused stem rot disease
117 of vanilla seedlings (Suprapta and Khalimi, 2009). Hada and Sharma (2017) prepared the
118 effective bioformulation of Cassia fistula plant extract with the combination of neem oil cake
119 and cow dung. Such type of bioformulation was found very effective to control early blight of
120 potato caused by Alternaria solani. Parveen and Sharma (2015) reported the significant control

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121 of ginger rot after treatment of bioformulation which prepared from leaf extract of Terminalia
122 arjuna along with cow dung slurry.
123 Hence, the present study focused on the efficacy of bioformulation of Lawsonia inermis against
124 the leaf spot disease of maize.
125
126 Materials and methods
127 Test crop and fungus
128 Curvularia lunata was isolated from infected leaves of maize collected from agriculture field of
129 Rajasthan College of Agriculture (RCA), Maharana Pratap University of Agriculture and Plant
130 Technology (MPUAT), Udaipur and nearby fields of Udaipur. The fungus was isolated from
131 infected leaves of maize by agar plate method (Horn, 2005). For this, infected leaves showing
132 typical symptoms of curvularia leaf spot were cut into small pieces and surface sterilized with
133 0.1% mercuric chloride (HgCl2) for two minutes. Then, they were washed thrice in distilled
134 water and transferred on potato dextrose agar (PDA) plates. The plates were incubated at 28 ±
135 20ºC for 6-7 days. Further, subcultures were made from the periphery of mycelia growth. These
136 cultures were stored at 4ºC and used in further study.
137
138 Pathogenicity test
139 Pathogenicity of the isolate, C. lunata was studied by spray inoculating pot grown plant of
140 maize. Surface sterilized (0.1 % HgCl2 for 2 min.) seeds were sown in pot. Mass multiplication
141 of the culture was done on PDA for 7 to 10 days at 28 ±2ºC in bio-oxygen demand (BOD)
142 incubator for preparation of spore suspension. The final concentration of the spore of suspension
143 was prepared as 1.0 × 106 conidia/ml. This spore suspension was sprayed on 50 days old plants
144 at the rate of 30 ml per plant. The inoculated plants were kept in humid chamber for 24 h and
145 then transferred to high humidity cage, where humid conditions were maintained by frequent
146 irrigations. The irrigation was done by spraying of plants with water. Initiation of infection was
147 visible as small light chloratic spot or flecks after 48 h after inoculation and characteristic
148 symptoms appeared within 7-10 days after inoculation. Re-isolation of pathogen was again done
149 to confirm the existence of the same pathogen (Supplementary Figure 1).
150

151 Preparation of crude extracts

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152 Fresh leaves of Lawsonia inermis were collected from the hedges on the campus of University
153 College of science, M.L.S. University, Udaipur. The plant was submitted for identification at
154 Herbarium of Department of Botany, University of Rajasthan, Jaipur, India. Plant was identified
155 as L. inermis vide voucher specimen number RUBL211447. The collected plant material was
156 shade dried at room temperature. It was then ground in an electrical grinder. The ground material
157 was passed through a sieve of mesh with size 60 to obtain a fine powder. It was then used to
158 prepare the extract. Crude extract was processed according to the cold extraction method
159 (Shadomy and Ingraff, 1974). Cold extraction was prepared in water, 50% alcohol and absolute
160 alcohol. 20 g of dried and powdered plant material was suspended in 100 ml water, 100 ml of
161 50% alcohol and 100 ml absolute alcohol separately, and kept for 48 h. After 48 h each mixture
162 was filtered through Whatman filter paper no. 1 and filtered material was vacuum dried using
163 rotary vacuum evaporator. The dried residue was used as extract and obtained solvent was reused
164 for other purposes.
165
166 Preparation of partially purified extracts
167 Successive separations of diverse partially purified organic components existent in dried plant
168 material were carried out by Reflux method of solvent extraction/hot extraction method
169 (Harborne, 1984; Kokate et al., 1990). Solvent sequence used for successive separation and there
170 chemical steps are as per the following sequence:
171 Petroleum ether → Benzene → Chloroform → Acetone → Alcohol → Methanol → Water
172 This method includes continuous extraction of dried plant material in soxhlet apparatus using a
173 sequence of organic solvents. Every time before extracting with next solvent the plant material
174 was dried in an oven at temperature up to 50ºC. 40 g dry plant material was kept in soxhlet
175 extraction unit and extracted with 280 ml petroleum ether till all petrol soluble fractions were
176 extracted. Residues were dried and used further for extraction with next solvent.
177 Antifungal activity of all extracts (Crude and partially purified) against C. lunata was observed.
178 Out of crude and partially purified extracts, 100% alcoholic extracts and acetone extracts showed
179 best activities, respectively (Barupal and Sharma, 2017a, b). These two extracts were further
180 used for preparation of formulations.
181
182 Inoculum development

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183 Potato dextrose agar (PDA) medium was used for culture of C. lunata. The inoculated petriplates
184 were incubated at 25ºC for seven days. Pure culture thus obtained was used for mass culture of
185 C. lunata in 250 ml flasks containing 25 ml of autoclaved potato dextrose broth. Each flask was
186 inoculated with 6 mm diameter of the fungus taken from the margins of a week old culture of C.
187 lunata, grown on PDA medium. The flasks were incubated at 25 ± 2ºC for 72 h. 25 ml of C.
188 lunata suspension (1.0 × 106 conidia/ml) per pot of soil was used as inoculum for
189 experimentation.
190
191 Preparation of pots and soil
192 Pots (33 cm height × 30 cm top diameter × 23 cm bottom diameter) were sterilized with 20%
193 CuSO4 solution whereas soil was autoclaved and then cooled. Sterile soil (10 kg per pot) was
194 mixed with inoculums and filled in the pre-sterilized pots.
195
196 Preparation of herbal formulations and standard solution
197 In vivo study of the preventive/protective action of the herbal formulation was based on results of
198 in vitro studies. Herbal formulations were prepared by using L. inermis leaf extracts, powder,
199 elicitor and binder in different ratio. 100% of clove bud oil cake and 100% of cow dung were
200 mixed with 100% alcoholic crude, partially purified acetone extract and fruit pulp powder. All
201 ingredients of the herbal formulation were used in the following combinations:
202 • Crude extract + elicitor + binder;
203 • Partially purified extract + elicitor + binder;
204 • Leaf powder + elicitor + binder
205 Here, we used clove bud oil cake as an elicitor which enhances the activity of extract
206 against the pathogen. We also used cow dung as a binder which binds elicitor and extract to form
207 a cohesive formulation. Mancozeb (a dithiocarbamate non-systemic agricultural fungicide with
208 multisite, protective action on contact) was used as standard antifungal and 10 mg/ml
209 concentration was prepared in sterile water.
210
211 Treatments and experimental design
212 The study was conducted during July 2017 to December 2017 in the Botanical Garden,
213 University College of Science, Mohanlal Sukhadia University, Udaipur, Rajasthan, India. Seed

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214 dipping method and foliar spray were used for the study of the preventive effect of the herbal
215 formulation (Jan et al., 2003; Ganie et al., 2013). On the basis of results obtained from in vitro
216 studies using poison food technique (Groover and Moore, 1962), we obtained four treatments out
217 of thirty treatments (Supplementary Table 1 & Supplementary Figure 2A, B) and also three
218 treatments were applied by mixing of Lawsonia leaf powder, clove bud oil cake, and cow dung
219 in different combinations. These treatments and combinations are as follow:
220 T1: Seeds were treated with formulation no. 4 [100% alcoholic crude extract (4 ml): 100% clove
221 bud oil cake (4 ml): 100% cow dung (2 ml)].
222 T2: Seeds were treated with formulation no. 12 [100% alcoholic crude extract (2ml): 100% clove
223 bud oil cake (6 ml): 100% cow dung (2 ml)].
224 T3: Seeds were treated with formulation no. 19 [Partially purified acetone extract (4 ml): 100%
225 clove bud oil cake (4 ml): 100% cow dung (2 ml)].
226 T4: Seeds were treated with formulation no. 28 [Partially purified acetone extract (3 ml): 100%
227 clove bud oil cake (4 ml): 100% cow dung (3 ml)].
228 T5: Seeds were treated with Lawsonia leaf powder (60 gm): clove bud oil cake (20 gm): cow
229 dung (20 gm).
230 T6: Seeds were treated with Lawsonia leaf powder (40 gm): clove bud oil cake (30 gm): cow
231 dung (30 gm).
232 T7: Seeds were treated with Lawsonia leaf powder (30 gm): clove bud oil cake (30 gm): cow
233 dung (40 gm).
234 Healthy seeds dipped in respective herbal formulations were planted in pre-sterilized soil pots
235 containing 15 kg soil infested with C. lunata inoculums. Four different controls were also
236 maintained respectively. These were as follows:
237 C1: seeds were treated with 10 mg/ml concentration of mancozeb sown in soil inoculated with C.
238 lunata.
239 C2: Untreated healthy seeds sown in unsterilized and uninoculated soil.
240 C3: Untreated healthy seeds sown in a sterilized soil inoculated with C. lunata.
241 C4: Untreated healthy seeds in sterilized uninoculated soil
242
243 Phytotoxicity of herbal formulations on seed germination of maize

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244
The percent seed germination of seeds treated with different herbal formulations (1 to 7) was
245
calculated using the Standard Blotter Method (SBM) established by the International Seed
246
Testing Association (ISTA). The randomly selected 7 sets of 30 seeds were surface sterilized
247
with 2% sodium hypochlorite for one minute and carefully rinsed 2-3 times with deionized
248
water. After this, each set of seeds were treated with different bioformulations for 30 minutes.
249
Each set of treated seeds were plated at the rate of 10 seeds per plate. Moist blotter paper was
250
placed in sterilized petriplates to maintain requisite moisture. Subsequently, seeds were placed
251
on it and covered with a lid. Petriplates were incubated at 27 ± 2ºC. One set of untreated seeds
252
was used as water control. After one week the petriplates were screened and percent seed
253
germination was calculated using the following formula:
Total number of seeds germinated
254 Percent seed germination (%) = ×100
Total number of seed plated
255
256 Effect of bioformulations on plant growth
257 Growth parameters like percent disease index (PDI) and percent efficiency of disease control
258 (PEDC), various growth characteristics such as plant height, number of leaves per plant, total
259 carbohydrate and protein content were recorded by conducting pot experiments.
260
261 Experimental outline
262 Four seeds were planted per pot at a depth of 5 cm. After 50 days first foliar spray of inoculum
263 (30 ml) of test fungus was done and after four days it was again repeated. Bioformulation and
264 fungicide treatment were given as post inoculation spray i.e. fungicidal and formulations sprays
265 were given 48 h after inoculation with the fungi, C. lunata. Observation after 10 days of the last
266 spray for PDI, PEDC, plant height, number of leaves per plant, total soluble sugar content and
267 total protein content were recorded from each pot again with the comparative study of positive
268 controls. Recorded data were subjected to analysis of CD and CV value. Three replicates were
269 maintained with each experiment (El-Mougy and Abdel-Kader, 2009; Mirkarimi et al., 2013). In
270 each replication four plants per pots were placed. The experiments were repeated three times
271 with three pots in each experiment.

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273 Disease rating and percent disease index
274 The percent disease index was recorded on 1 to 5 standard disease rating scale as recommended
275 by Directorate of Maize Research, New Delhi proposed in All India Coordinated Maize
276 Improvement Project Workshop, 2011. Detailed of the disease rating scale and PDI are
277 mentioned as below:
278 Rating scale for leaves
Percent leaf area infected Score
1-20 % leaf area infected 1
21-40 % leaf area infected 2
41-60 % leaf area infected 3
61-80 % leaf area infected 4
81-100 % leaf area infected 5
279
280 The percent disease was calculated using the following formula:
Sum of all individual disease ratings
281 Percent Disease Index (PDI) = × 100
Total numbers of leaves assessed × Maximum rating
282
283 Hence, the disease severity was recorded after 10 days of inoculation on the standard rating scale
284 (1-5) and the PEDC was determined by using the formula given by Chester (1959) and Wheeler
285 (1969).
PDI in control - PDI in treatment
286 Percent Efficacy of Disease Control ( PEDC ) = × 100
PDI in control
287
288 Estimation of chlorophyll (Chl a, Chl b and total Chl) and total carotenoids contents
289 The photosynthetic pigments were extracted and determined according to the methods of
290 Metzner et al. (1965) and Meena et al. (2016a). 1.0 gm of plant leaf tissues were weight and
291 chopped into small pieces in a clean mortar and pestle. Leaves tissue were homogenized to a fine
292 pulp with the addition of 85% acetone for 5 min. The homogenates were centrifuged, and the
293 supernatants were made up to 30 ml with 80% acetone. Absorbance was measured at 470 nm,

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294 645 nm and 663 nm for total carotenoids content, chlorophyll a, b and total chlorophyll against a
295 blank of aqueous acetone.
296 The concentration of the pigment fractions (Chl a, Chl b and total chlorophyll) and total
297 carotenoids were calculate using to the Lichtenthaler and Wellburn (1983) reported equations.
298
299 Determination of total carbohydrate (soluble sugar) contents
300 Estimation of carbohydrate content was carried out by the method of Yemm and Willis (1954).
301 The leaves samples were oven dried and powdered in a grinder. 0.5 gm of powdered sample was
302 homogenized in 2 ml of 80% ethanol. The total volume was made up to 10 ml. The homogenate
303 was subsequently centrifuged for 10 minutes at 3,000 rpm. The supernatant solution was used for
304 analysis. A suitably diluted aliquot was mixed with 4.5 ml of freshly prepared anthrone reagent
305 and heated for 10 min. The percent transmission was noted on a spectrophotometer at 620 nm.
306 All the samples were replicated thrice. The quantity of the total water soluble carbohydrates was
307 calculated with the help of standard curve and was expressed as mg g-1 dry weight of samples.
308
309 Determination of soluble protein contents
310 The soluble protein contents were estimated according to the method suggested by Bradford
311 using Coomassie Brilliant Blue (CBB) G-250 (Bradford, 1976). 50 mg of fresh plant material
312 was homogenized with phosphate buffer (0.2 M, pH 6.2) and the homogenate was consequently
313 centrifuged for 10 minutes at 10,000 rpm. The supernatant was separated and the volume was
314 made up to 10 ml. One ml of this protein extract was added to 5 ml of CBB reagent and vortexed
315 for 30 seconds for developing colour which develops within 2 minutes. The percent
316 transmittance was noted with a spectrophotometer at 595 nm, using the pure reagent as blank. A
317 standard curve using 10-100 µg ml-1 protein from a stock solution of 100 µg ml-1 bovine serum
318 albumin was plotted.
319
320 Statistical analysis
321 All the statistical analysis and calculation were accomplished by using IBM SPSS Statistics Ver.
322 20 software. The statistical data were expressed as the mean of three independent replications ±
323 standard error (SE) of at least three replicates of each experiment. Also, we have tested these
324 replicates through the coefficient of variation followed by range test at P ≤ 0.05 significance

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325 level which shows the consistency among these. The experiments were performed in triplicates
326 and all the experiments repeated twice via totally randomized design.
327
328 Results and observations
329 Isolation and morphological identification of plant pathogenic fungus
330 Cultural characteristics of isolate were investigated by growing them on PDA medium at 28 ±
331 2ºC. The sporulating culture was identified on the basis of colony morphology, conidial
332 characteristics, and morphological characters of somatic and reproductive structures (Fig. 1) by
333 using Olympus microscope (Olympus, Japan, Model No. BX51). Conidiophores were erect,
334 unbranched, septate, flexuose in the apical portion with flat and brown scars. Conidia were
335 smooth-walled, olivaceous brown, end cells slightly paler; conidia obovoidal to predominantly
336 clavate, curved at the subterminal cell, 20-34 × 7.5-13.5 µm, 1-3-septate, the subterminal cell
337 swollen and distinctly larger than the remaining cells. Identification of fungus was also done by
338 the senior maize pathologist (AICRP-Maize, New Delhi) professor S. S. Sharma, Department of
339 Plant Pathology, RCA, MPUAT (Certificate of identification of C. lunata given in
340 Supplementary Data Sheet 1). Maize grains were procured from the local market of Udaipur and
341 used for experiments.
342
343 Percent disease index (PDI) and percent efficiency of disease control (PEDC)
344 Results of the effect of herbal formulations on PDI (Fig. 2) and PEDC are listed in
345 Supplementary Table 1. PDI and PEDC were documented to determine the efficacy of
346 bioformulations against leaf spot disease of maize. A substantial control of leaf spot disease was
347 recorded with bioformulations treatment T3, T4, T2 and T1 as compared to others and its PDI
348 and PEDC were recorded 9.10%, 10.00%, 17.50%, 19.00% (Fig. 2) and 89.47%, 88.43%,
349 79.76%, 78.03% (Supplementary Table 2), respectively. Statistical analysis at (P≤ 0.05) CD
350 revealed that all the treatments are significant.
351
352 Number of Leaves/plant
353 Results of the effect of bioformulations on numbers of leaves/plant are given in Figure 3. The
354 data indicate that slight reduction in the number of leaves/plant due to infection of the pathogen.
355 However, significant increases in the number of leaves/plant were observed due to treatment

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356 with herbal formulations as well as mancozeb. The maximum increase in the number of
357 leaves/plant was observed with bioformulations treatment T3 (11.00) followed by T4 (10.00), T2
358 (9.50), T1 (9.50), T5 (9.00), and T6 (8.15), respectively. Lowest number of leaves/plant was
359 observed in T7 (7.00). Statistical analysis at (P ≤ 0.05) CD revealed that all the treatments are
360 significant.
361
362 Plant height
363 Results of the effect of bioformulations on plant height are given in Figure 4. The data indicate
364 that a slight reduction in plant height due to infection. A significant increase in plant height was
365 observed due to treatment with herbal formulations. Maximum plant height was observed in
366 bioformulations treatment T3 (177.23 cm) followed by T4 (170.26 cm), T2 (168.40 cm), T1
367 (166.63 cm), T5 (165.26 cm), and T6 (163.43 cm), respectively. The least plant height was
368 observed in T7 (161.46 cm). Statistical analysis at (P ≤ 0.05) CD revealed that all the treatments
369 are significant.
370 However, 100% percent seed germination was observed for seeds treated with all
371 formulations which were at par with control i.e. 100%, but the seedlings growth was found
372 higher in T3 bioformulations treatment in comparison to other boiformulations treatment (Fig.
373 5). Figure 5 also showed the in vivo study of the preventive effect of herbal formulations which
374 indicate the T3 bioformulation treatment showed the best activity followed by T4, T2, T1, T5,
375 T6, and T7, respectively.
376
377 Biochemical analysis
378 Chlorophyll (Chl a, Chl b and total Chl) and carotenoids contents
379 A significant increase in chlorophyll contents was observed due to treatment with herbal
380 formulations. Maximum chl a content was observed in bioformulations treatment T3 (13.18 mg
381 gm-1) followed by T4 (11.04 mg gm-1), T2 (10.75 mg gm-1), T1 (9.40 mg gm-1), T5 (8.87 mg gm-
1
382 ), and T6 (7.92 mg gm-1), respectively. The least Chl a content was observed in T7 (7.05 mg
383 gm-1). Statistical analysis at (P ≤ 0.05) CD revealed that all the treatments are significant.
384 Chl b content was found highest in bioformulations treatment T3 (5.39 mg gm-1) followed by T4
385 (4.25 mg gm-1), T2 (3.96 mg gm-1), T1 (2.61 mg gm-1), T5 (2.08 mg gm-1), and T6 (1.13 mg gm-
1
386 ), respectively. The least Chl b content was observed in T7 (0.98 mg gm-1). In the same way, the

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387 total chlorophyll content followed the similar pattern and found to be maximally in
388 bioformulations treatment T3 (18.57 mg gm-1) followed by T4 (15.29 mg gm-1), T2 (14.71 mg
389 gm-1), T1 (12.01 mg gm-1), T5 (10.95 mg gm-1), and T6 (9.05 mg gm-1), respectively. The
390 minimum total chlorophyll content was observed in T7 (8.03 mg gm-1) (Table 1).
391 The total carotenoids content was also found highest in bioformulations treatment T3 (40.99 mg
392 gm-1) followed by T4 (38.71 mg gm-1), T2 (26.13 mg gm-1), T1 (23.43 mg gm-1), T5 (34.30 mg
393 gm-1), and T6 (32.47 mg gm-1), respectively and minimum in T7 (27.45 mg gm-1) (Table 1).
394
395 Carbohydrate content
396 The result of the effect of herbal formulations prepared from L. inermis leaf crude and partially
397 purified acetone extract, powder and clove bud oil cake on biochemical parameter i.e.
398 carbohydrate content of maize crop are summarized in Figure 6. Data clearly indicate that,
399 treatment with herbal formulations increasing carbohydrate content of the host plant. The
400 maximum carbohydrate content was observed with bioformulation treatment T3 (7.57 mg gm-1)
401 followed by T4 (7.07 mg gm-1), T2 (6.46 mg gm-1), T1 (6.16 mg gm-1), T5 (5.36 mg gm-1), T6
402 (5.12 mg gm-1), and T7 (5.07 mg gm-1), respectively. Statistical analysis at (P ≤ 0.05) CD
403 revealed that all the treatments are significant.
404
405 Protein contents
406 The result of the effect of bioformulation on protein contents was shown in Figure 7. The protein
407 contents were increased as PDI is increased. Bioformulation treatment T7 (1.34 mg gm-1)
408 showed maximum protein content i.e. followed by T6 (1.24 mg gm-1), T5 (1.20 mg gm-1), T1
409 (1.11 mg gm-1), T2 (1.08 mg gm-1), and T4 (0.96 mg gm-1), respectively. The minimum protein
410 content was observed in T3 (0.95 mg gm-1). Statistical analysis at (P ≤ 0.05) CD revealed that all
411 the treatments are significant.
412
413 Discussion
414 Worldwide, maize is an essential food crop but it is susceptible to various bacterial, viral and
415 fungal diseases. In maize, C. lunata causes leaf spot disease which alone affects the yield
416 damage by up to 60% of the total production (Fu-Hua et al., 2006; Akinbode, 2010; Bisht, 2016;
417 Meena et al., 2016b, c). In the current examination, we report for the first time the efficacy of

14
418 bioformulation (Combined application of plant extracts, elicitor, and binder) to persuade the
419 defense reactions against leaf spot disease in maize and to stimulate sustainable plant growth
420 under natural environment. Many strategies have been applied to regulate the leaf spot disease of
421 maize produced by C. lunata using chemicals and other bio-agents but there is no report on the
422 evaluation of such kind of bioformulation against this disease in maize. A few reports are
423 available related to such kind of integrated approach for other plant diseases. Hada and Sharma
424 (2017) investigated the effective bioformulation as Cassia fistula plant extract and its
425 combination with neem oil cake and cow dung. Such type of bioformulation was found to be
426 very effective in control of early blight of potato caused by fungus Alternaria solani. Parveen
427 and Sharma (2015) reported the significant control of ginger rot after treatment with
428 bioformulation (prepared from Terminalia arjuna leaf extract along with cow dung slurry). PDI
429 and PEDC are primary important parameter to find out the assessment of any disease (Chester,
430 1959; Wheeler, 1969; Gopinath et al., 2006; Reddy et al., 2013; Meena et al., 2017b, c, d; Wani
431 et al., 2017). In the present study, PDI and PEDC were recorded in pot experiment to determine
432 the efficacy of bioformulations against leaf spot disease of maize. Whenever a novel chemical or
433 formulation is prepared for use as a control agent, it is necessary to check it for toxicity to host
434 plant. Only then it is applied for field trials. Toxicity studies can be done by various methods.
435 Most common study of the effect of the formulation on seed germination by Standard Blotter
436 Method (Mehta and Sharma, 2017; Masangwa et al., 2017; Meena et al., 2017e, f) toxicity of the
437 formulation was evaluated by seeing its effect on percent seed germination by this method. 100%
438 seed germination after all treatments indicates that the bioformualtion has no toxic effect on seed
439 germination and can be safely used in field trials.
440 Among all the treatments, a significant in vivo control of leaf spot disease was recorded
441 with bioformulation treatment T3 followed by T4, T2, and T1. Hence, further studies can be
442 conducted using this combination. The defense response of bioformulation might be due to direct
443 activities such as (a) through membrane damage, and (b) positively influences the transcription
444 and translations processes, and prevent the fungal proliferation (Akila et al., 2011; Senthilraja et
445 al., 2013). On another side, indirect activity might be exerted by bioformulation through
446 stimulated plant immune response by enhanced activities of antioxidant and defense enzymes
447 (Valueva et al., 2001; Supuran et al., 2002; Valueva and Mosolov, 2004; Christeller, 2005;

15
448 Mosolov and Valueva, 2005; Habib and Fazil, 2007; Meena et al., 2017g; Meena and Samal,
449 2019).
450 To take the benefit of growth promoting consequence of bioformulations, maize seeds
451 were treated with different bioformulations followed by foliar spray in the pot experiment. Plant
452 extracts based bioformulations considerably enriched the growth development of maize plants in
453 pot experiments as compared to control and fungicide treatments. Statistical analyses showed
454 that in bioformulation number T3, notably increased in plant height, number of leaves per plant,
455 chlorophyll and carotenoids contents, total soluble sugar content was observed followed by
456 formulation number T4, T2, and T1 (Figs. 2-6 & Table 1). However, total soluble protein content
457 was observed to be less as compared to control except mancozeb control (C1). Reason might be
458 synthesis of PRP in control plants due to fungal infection (De Tapia et al., 1986; Tuzun et al.,
459 1989; Ye et al., 1990; Meena et al., 2017h), Whereas increased sugar content in treated plant can
460 be directly correlated to enhanced photosynthesis which in turn affect the growth of plant (Qazi
461 et al., 2012; Sui et al., 2015). Several researchers have used various kind of bioformulations like
462 PGPR based bioformulation, nano-particle based bioformulations (Rangeshwaran and Prasad,
463 2000; Srivastava et al., 2001; Chattopadhyay et al., 2001; Jana et al., 2001; Goel et al., 2002;
464 Anjaiah et al., 2003; Ansari, 2005; Altindag et al., 2006; Kumar et al., 2007; Akhtar and
465 Siddiqui, 2007, 2008; Usharani et al., 2009; Khare and Arora, 2010; Hameeda et al., 2010; Khare
466 et al., 2011; Senthilraja et al., 2013; Choudhary et al., 2017; Kumari et al., 2018a, b; Meena and
467 Swapnil, 2019) but these approaches are very costly and time consuming as compared to the
468 present approach of using a combination of plant extract, elicitor and binder.
469
470 Conclusion
471 Since, demand of food/feed crop free from synthetic constituents has exponentially increased in
472 current years in the direction of rancid harmful effects of synthetic supplements and to avoid the
473 enlargement of resistance in pathogens, a novel approach is imperative to be amended to
474 reinforce plants innate immunity to survive with mutating plant pathogens, diminish chemical
475 use together with sustained plant growth. Bioformulations have been established as a promising
476 plant protection and growth development promontory agent in our earlier and ongoing
477 investigations. Its exceptional capacity to sustain the plant growth development under disease
478 conditions makes it an extremely viable, effective and functional agent. These bioformulations

16
479 could be significant in the direction of sustainable agriculture without destroying biological
480 system. The manufactured bioformulations have enormous potential to be economically
481 investigated for agriculture use.
482
483 Conflict of interest
484 The authors declare that there is no conflict of interest.
485
486 Acknowledgments
487 The authors are thankful to Professor S. S. Sharma, maize pathologist (AICRP-Maize, New
488 Delhi), Department of Plant Pathology, RCA, MPUAT, for helping in the identification of
489 fungus. The author TB is grateful to University Grants Commission (UGC), New Delhi,
490 Government of India, for the award of UGC-BSR research fellowship. The author MM is
491 thankful to University Grants Commission (UGC), New Delhi, Government of India, for the
492 award of UGC-Startup Research Grant (UGC Faculty Research Promotion Scheme; FRPS)
493 which is sustained by Mohanlal Sukhadia University, Udaipur, Rajasthan, India.
494
495 Author’s contributions
496 TB: Planned and designed the experimental work, performed the experiments, wrote and edited
497 the manuscript. MM: Analyzed all the data, results assessment and evaluation, implemented all
498 the statistical analysis, prepared and wrote the manuscript, also reviewed and edited the final
499 version of the manuscript. KS: Supervised the entire work. The final version of the manuscript
500 read and approved by all the authors.
501
502

17
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28
775 Figure legends

776 Figure 1. Cultural and morphological characteristics of Curvularia lunata. (A) Infected leaves;
777 (B) C. lunata colony on potato dextrose agar (PDA) medium plate; and (C) Mycelium and
778 conidia of C. lunata (at 400X).

779 Figure 2. Comparative efficiencies of bioformulations against leaf spot disease of maize in pot
780 culture. The statistical data were expressed as the mean of three independent replications ±
781 standard error (SE) of three replicates of the experiment. SE is shown as a thin linear bar. Data
782 showed critical difference (CD = 0.16), and coefficient of variation (CV = 0.29) followed by range
783 test at the P ≤ 0.05 significance level.

784 Figure 3. Effects of treatments on the number of leaves/plant of maize. The statistical data were
785 expressed as the mean of three independent replications ± standard error (SE) of three replicates
786 of the experiment. SE is shown as a thin linear bar. Data showed the critical difference (CD = 0.46),
787 and coefficient of variation (CV = 4.35) followed by range test at the P ≤ 0.05 significance level.

788 Figure 4. Effects of treatments on the plant height (cm) of maize. The statistical data were
789 expressed as the mean of three independent replications ± standard error (SE) of three replicates
790 of the experiment. SE is shown as a thin linear bar. Data showed critical difference (CD = 0.43),
791 and coefficient of variation (CV = 0.22) followed by range test at the P ≤ 0.05 significance level.

792 Figure 5. (A) Phytotoxicity of herbal formulations on seed germination of maize; (B) In vivo
793 study of the preventive effect of herbal formulations.

794 Figure 6. Effects of treatments on the total carbohydrate of maize. The statistical data were
795 expressed as the mean of three independent replications ± standard error (SE) of three replicates
796 of the experiment. SE is shown as a thin linear bar. Data showed critical difference (CD = 0.08),
797 and coefficient of variation (CV = 0.962) followed by range test at the P ≤ 0.05 significance
798 level.

799 Figure 7. Effects of treatments on the total protein contents of maize. The statistical data were
800 expressed as the mean of three independent replications ± standard error (SE) of three replicates

29
801 of the experiment. SE is shown as a thin linear bar. Data showed critical difference (CD = 0.04),
802 and coefficient of variation (CV = 2.20) followed by range test at the P ≤ 0.05 significance level.

30
803 Table 1: Estimation of chlorophyll contents (Chl a, b and total Chl), and total carotenoids contents in maize applying different
804 bioformulation against leaf spot disease.
Treatments Combinations Chl a Chl b Total chl Total
(mg gm-1) (mg gm-1) (mg gm-1) carotenoids
(mg gm-1)
T1 Seeds were treated with 9.40 ± 0.24def 2.61 ± 0.70c 12.01 ± 1.44cd 32.47 ± 2.84c
formulation no. 4 [100% alcoholic
crude extract (4 ml): 100% clove
bud oil cake (4 ml): 100% cow
dung (2 ml)]
T2 Seeds were treated with 10.75 ± 1.30ef 3.96 ± 0.91d 14.71 ± 2.74de 34.30 ± 2.44cd
formulation no. 12 [100% alcoholic
crude extract (2ml): 100% clove
bud oil cake (6 ml): 100% cow
dung (2 ml)]
T3 Seeds were treated with 13.18 ± 1.04g 5.39 ± 0.72e 18.57 ± 3.03f 40.99 ± 3.99e
formulation no. 19 [Partially
purified acetone extract (4 ml):
100% clove bud oil cake (4 ml):
100% cow dung (2 ml)]
T4 Seeds were treated with 11.04 ± 1.84f 4.25 ± 0.84d 15.29 ± 2.60def 38.71 ± 2.82de
formulation no. 28 [Partially
purified acetone extract (3 ml):
100% clove bud oil cake (4 ml):
100% cow dung (3 ml)]
T5 Seeds were treated with Lawsonia 8.87 ± 1.04cde 2.08 ± 0.51bc 10.95 ± 1.76bc 27.45 ± 2.74b
leaf powder (60 gm): clove bud oil
cake (20 gm): cow dung (20 gm)

31
 T6 Seeds were treated with Lawsonia 7.92 ± 1.32bcd 1.13 ± 0.42ab 9.05 ± 1.65abc 26.13 ± 1.84b
leaf powder (40 gm): clove bud oil
cake (30 gm): cow dung (30 gm)
T7 Seeds were treated with Lawsonia 7.05 ± 1.54abc 0.98 ± 0.31a 8.03 ± 1.01ab 23.43 ± 1.23b
leaf powder (30 gm): clove bud oil
cake (30 gm): cow dung (40 gm)
C1 Seeds were treated with 10 mg/ml 11.34 ± 1.74fg 4.55 ± 0.80de 15.89 ± 2.74ef 37.31 ± 3.74de
(Mancozeb) concentration of mancozeb sown in
soil inoculated with C. lunata
C2 Untreated healthy seeds sown in 6.40 ± 0.67ab 0.84 ± 0.05a 7.24 ± 0.96a 24.66 ± 1.98b
unsterilized and uninoculated soil.
C3 Untreated healthy seeds sown in a 5.80 ± 0.54ab 0.74 ± 0.04a 6.54 ± 0.84a 22.96 ± 1.32b
sterilized soil inoculated with C.
lunata
C4 Untreated healthy seeds in 5.23 ± 0.44a 0.62 ± 0.03a 5.85 ± 0.77a 18.27 ± 1.04a`
sterilized uninoculated soil
805
806 Note: the results are expressed as the mean of three replicates and “±” indicate the SE of the mean. Different letters indicate that the
807 values are significantly different from each other (P ≤ 0.05).
808
809

32
Highlights

• The efficacy of bioformulations to induce the defense responses against leaf spot disease in

maize.

• A significant control of leaf spot disease was recorded with bioformulations T3, T4, T2, and

T1 as compared to others.

• Total soluble protein content was observed to be less in T3 as compared to control.

• Plant height, number of leaves per plant, chlorophyll, carotenoids and total soluble sugar

contents are increased remarkably in T3 bioformulation treated plants.

• The synthesized bioformulations have immense potential to be commercially explored for

agriculture use.

• These bioformulations can be used to promote sustainable plant growth under field

conditions.
Author’s contributions statement
TB: Planned and designed the experimental work, performed the experiments, wrote and edited
the manuscript.
MM: Analyzed all the data, results assessment and evaluation, implemented all the statistical
analysis, prepared and wrote the manuscript, also reviewed and edited the final version of the
manuscript.
KS: Supervised the entire work.
The final version of the manuscript read and approved by all the authors.