Effect of Soil Improvement Practices and NAA Application on Yield and Phytophthora Diseases of Sweet Pepper (Capsicum annuum L.)

Introduction

Capsicum annuum L. belongs to the family Solanaceae and has attained an important status and special significance in the mid-hills of Himachal Pradesh where it is cultivated as an off-season crop during the summer months. Bell pepper is a high-value crop worldwide in recent years and occupies a proud place among warm-season vegetables in international cuisine because of its delicacy, pleasant flavour, and great nutrient content [1]. The significant problems caused by soil-borne pathogens in crop production worldwide include reduced crop performance, decreased yield, and higher production costs [2].

The first description of Phytophthora capsici was given by L H Leonian as a pathogen of chili pepper in the year 1920 [3]. P. capsici causes foliar blightning, fruit rot, collar rot, damping-off, and wilting of susceptible hosts like cucurbits and peppers [4] and causes great economic losses. Management of P. capsici disease is limited due to the long-term survival of the pathogen as oospores in the soil [5], a wide host range [6], a moment of the pathogen in surface water used for irrigation and so many [7].

Rainfall is the most influential environmental factor affecting the incidence and progress of this disease, and flowing water appears necessary for the detachment and spread of sporangia [8].  When heavy rainfall occurs, disease severity may develop even in well-drained soil as pathogens dispersed by wind-driven rain, and the flow of runoff water may distribute inoculums down rows [9 &4], especially in flat beds. Crop plantation on the raised surface can prevent soil moisture accumulation at the plants’ base. Covering the raised beds with plastic mulch covers is desirable to increase yield by reducing the crop loss by Phytophthora capsici [10 &11]. Water management has been reported as an important strategy in managing soil-borne diseases like Phytophthora [12]. As the Bell pepper crop is sensitive to water logging, in the such case raised beds offer the potential to reduce water logging stress through improved surface drainage, and check the spread of soil-borne diseases like Phytophthora [13-15].  Soil drainage by preparing raised surfaces and furrows should be designed to prevent the formation of standing water; this will help to reduce the amount and duration of contact between fruit and wet soil [16]. 

The use of mulching is another way to restrict the spread of P. capsici infection. The word mulch has been derived from the German word molsch means “easy to decay,” and mulches have widely been used for vegetable production since ancient times [17]. Mulching minimizes soil moisture losses [18-21], water runoff, weed population, nutrient loss, and soil erosion and enhances crop yield [22 &23], improving the infiltration capacity of the soil, and soil temperature regulation. Plastic mulch either it is black or silver mulch might help reduce water splashing and thus turn the spread of Phytophthora inoculums [16]. Mulches act as the beating action of raindrops that carry zoospores of Phytophthora or other soil-borne diseases. These spores attach themselves to foliage, lower fruits, and shoots of the plant which causes secondary infection of disease [24]. Mulches help in nitrifying many beneficial microorganisms which compete with the pathogenic spores or sometimes release chemicals for the inhibition of pathogens [25].

NAA is a synthetic plant hormone in the auxin family, which has been used for a long time to improve the quality and quantity of fruits and vegetables [26]. The application of NAA increases most of the growth and yield characteristics [27]. Metabolism of auxin generates the energy-rich phosphate and precursor of many metabolic processes, which leads to growth initiation, and such condition favours increased yield as most of the assimilates translocated from source to sink under a favourable environment [28]. NAA also plays a great role in the suppression of diseases like Phytophthora, it reduces mycelium growth rate [29]. Circulation of NAA is very slow in plant tissues than other auxins, and the flux transport rate of NAA is also slow. So, when NAA is applied exogenously on plant tissues, it accumulates in the upper layer of cells where it initiates resistance against the fungus for a short time period [30]. In considering the aforementioned, a research study was carried out to assess the impact of various soil improvement strategies on bell pepper yield and phytophthora disease.

Materials and Methods

Field experiments to determine the effect of soil improvement practices and Naphthalene acetic acid on yield and Phytophthora diseases of bell pepper were conducted in a randomized block design (factorial) during Khraif seasons (April to August) of 2017-18 and 2018-19 at Experimental Farm of the Department of Vegetable Science, Dr. YS Parmar University of Horticulture and Forestry, Nauni, Solan, HP.

The experiment comprised of two planting methods viz., P₁ (raised bed planting method) and P₂ (flat bed planting method), three levels of mulch materials viz., M₁ (black polythene mulch), M₂ (silver/black polythene mulch) and M₃ (no mulch) and two NAA levels viz., N₁ (application of NAA @ 15ppm at 30 and 45 days after transplanting) and N₂ (no NAA application) (Table 1). Thus, there were 12 treatment combinations in all replicated thrice. The height of the raised beds was 15 cm above the ground level and two beds were separated by 45 cm distance. Seedlings were transplanted on well-prepared plots in April 2017 and 2018 at a spacing of 60×45 cm in a plot having dimensions of 1.20×9.45 m, accommodating 42 plants per plot. Mulches of 50µ (200 gauge thickness) were applied in plots according to the treatment combinations. After that holes were made in the mulch as per the recommended spacing of the plants.

Bell pepper cultivar “Solan Bharpur” was used in the present study. This cultivar was released by the Department of Vegetable Science, Dr. YSP UHF Nauni, Solan. It is a high-yielding open-pollinated variety which is a selection from segregating populations having four-lobed fruits. This variety has received a tremendous response from the capsicum-growing farmers of the state being a very popular variety well suited for the mid-hills of Himachal Pradesh.

Results and Discussion

It is clear from the data presented in Table 2 that there was a significant effect of different planting methods, mulches, and NAA levels on diseases and the yield of sweet pepper.

Planting methods

Lower leaf blight severity (2.52 %), the incidence of collar rot (5.43 %), and fruit rot (4.85 %) were recorded in those plants which were grown on raised beds, whereas, the plants which were grown on flat beds had higher severity of leaf blight (3.68 %), the incidence of collar rot (6.65 %) and fruit rot (6.27 %) caused by Phytophthora capsici. Similar plots observed higher increased yield per plot (46.46 kg) and per hectare (327.74 q). Phytophthora blight, root rot, and fruit rot are very serious problems in solanaceous vegetables. The main source of pathogens is infested soil. Pathogen survives in contaminated soil for many years [31]. Planting into raised beds can prevent soil moisture accumulation at the base of the plants. Sharma et al. [32] concluded that raised bed planting + Eucalyptus globulus (10 %) + Bavistin (0.1 %) was the most effective and significantly best treatment with the least disease incidence and maximum disease control in capsicum crop due to proper soil drainage in raised beds. Increased yield in raised bed planting could be due to improved crop growth due to better soil conditions, minimum tillage, and improved soil structure through increased organic matter level and root activity of pepper and pea [33-35]. Also, there was minimum compaction of the soil which resulted in reduced mechanical inputs and better aeration.

Mulch material

As regards mulches (Table 2), the minimum severity of leaf blight (1.55 %) was recorded in the plants M₁ which was statistically at par (1.63 %) with M_2, and the maximum (6.13 %) severity was recorded in M₃. Similarly, a lower (2.86 %) incidence of collar rot was recorded when the plants were grown using black mulch while a maximum (11.18 %) was recorded when the plants were grown without mulch. In the case of fruit rot, less (2.89 %) incidence of fruit rot was observed in M₂, which was statistically at par (2.93 %) with M₁ while more (10.85 %) incidence was calculated in no mulch plots. Silver mulched plots also produced increased yield per plot (49.93 kg) and yield per hectare (352.22 q), whereas, decreased yield (37.33 kg per plot) and (263.33 quintals per ha) was produced by the plants grown on bare soil. Plastic mulching reduces disease by preventing the formation of a bridge of dead tissues between the soil and the plants. It also reduces the weed population which leads to the starvation of fungus by elimination of weed hosts [36]. Studies conducted by Ashrafuzzaman et al. [37] and Verma et al. [38] revealed that the use of silver black plastic mulch significantly increased yield due to minimum weeds, better soil moisture retention, optimum root zone temperature, and better nutrient availability to the plants. Higher marketable fruit yield from mulched plots than the plants produced on bare soil was also reported in bell pepper [39 &40]. This difference could be attributed to mineral nutrient uptake in the mulched plots through improved root temperature.

NAA levels

In the case of NAA levels (Table 2), the plants which were sprayed with Naphthalene acetic acid had minimum severity of leaf blight, the incidence of collar rot and fruit rot i.e., (2.98 %), (5.86 %) and (5.40 %), respectively, while the plants with no NAA application recorded maximum (3.23 %) severity of leaf blight, collar rot (6.23 %) and fruit rot (5.72 %) incidence. Similarly, superior yield per plot (47.83 kg) and per hectare (337.45 q) was recorded in N₁, whereas, the lowest yield per plot (42.75 kg) and per hectare (301.56 quintals) was recorded in N₂. There is ample evidence that NAA is a potential antifungal agent [41-43], however, the role of auxin in the growth and development of fungi has not yet been clearly elucidated. NAA-treated plants remained physiologically more active to build up sufficient food material for developing a greater number of flowers and fruits, ultimately leading to more fruit setting and consequently more yield of better-quality fruits in chilli and sweet pepper [44-46]. Basuchaudhuri [26] noted a significant effect of Planofix (NAA) on plant height, number of fruiting branches, and yield probably due to functions of NAA like the stimulation of cell division, cell elongation, elongation of shoots, photosynthesis, RNA synthesis, membrane permeability, and water uptake in many physiological processes.

Consortium effect

The interaction of P, M, and N was found to be significant for all the diseases and yield characters (Table 3).  The minimum severity of leaf blight (1.13 %) was recorded in T₃, which was statistically at par (1.19 %) and (1.26 %) with T₁ and T₂, respectively (Fig. 1). Similarly lower incidence of collar rot (2.64 %) was recorded in T₇, which was at par (2.75 %) and (2.91%) with T₁and T₂, respectively (Fig. 2). Lower incidence of fruit rot (2.20 %) was recorded in P₁M₁N₁ (Fig. 3). This was at par (2.27 %) and (2.55 5) with P₁M₂N₁ and P₁M₂N_2, respectively. Control plots (P₂M₃N₂) recorded more severity of leaf blight (7.64 %), the incidence of collar rot (12.81 %), and fruit rot (12.31 %). Phytophthora is activated only in waterlogged soils. Wet soil triggers oospores to germinate and produce sporangia that release zoospores within thirty minutes. Zoospores are attracted to roots and infect root hairs. Sporangia produced above ground are splashed onto fruit and other plants in the field. When sporangia land on plant tissues, the disease starts quickly [47 &48]. Therefore, optimizing vertical drainage should effectively reduce the period of soil saturation and subsequent damage due to disease [49]. So, raised beds are a better option to reduce the harmful effect of soil-borne diseases like Phytophthora. Planting with mulches can reduce the splashing of P. capsici-infested soil onto plant material and be shown to lower disease incidence and pathogen spread as compared to bare soil [50 &16].

Raised bed planting + silver mulch + NAA application produced excellent yield (54.53 kg/ plot) and (384.69 q/hectare), whereas, lower yield per plot (34.23 kg) and per hectare (241.48 q) was produced by P₂M₃N₂ (Fig. 4 and 5). According to Islam et al. [51] planting the crop in raised bed create better drainage, conserved moisture during the dry season, greater soil depth, adequate soil moisture throughout the year, and increased water level above field capacity which are probable factors responsible for increased yield. Mulch surface colour that reflects light affected the growth of bell pepper plants by influencing the amount and quantity of upwardly reflected light in addition to modification of soil temperature [52&53]. Mohammad et al. [54] and Kumari et al. [55] reported the significant effect of NAA on plant processes like vascular tissue differentiation, root initiation, apical dominance, fruit set, and flowering. All these processes are directly or indirectly responsible for an increase in yield and yield contributing characters.

Based on the findings from this research, growing bell peppers in a raised bed with silver/black mulch and NAA @ 15 ppm at 30 and 45-days post-transplantation is the preferred way of improving crop yield and minimizing the incidence of Phytophthora disease.

Conflict of Interest: The authors declare that no conflict of interest exists

Acknowledgment: I extend my gratitude to the Dr. YS Parmar University of Horticulture and Forestry,Nauni, Solan, H.P. for providing financial support in the form of a scholarship during the course of study.

References

1. Agrawal A, Gupta S, Ahmed Z (2007) Influence of plant densities on the productivity of bell pepper (Capsicum annuum L.) under greenhouse in high altitude cold desert of Ladakh. Acta Hortic756: 309-14.
2. Panth M, Hassler SC, Baysal-Gurel F (2020) Methods for management of soilborne diseases in crop production. Agri 10: 16.
3. Leonian LH (1922) Stem and fruit blight of peppers caused by Phtopthora capsica sp. Nov. Phytopath 12: 401-08.
4. Hausbeck MK, Lamour KH (2004) Phtopthora capsica on vegetable crops: Research progress and management challenges. Plant Dis 88: 1292-1303.
5. Lamour KH, Hausbeck MK (2003) Effect of crop rotation on the survival of Phytophthora capsica in Michigan. Plant Dis 87: 841-45.
6. Erwin DC, Ribeiro OK (1996) Phytopthora diseases worldwide. American Phytopathological Soc, St. Paul, MN, USA.
7. Granke LL, Windstam ST, Hoch HC, Smart CD, Hausbeck MK (2009) Dispersal and movement mechanisms of Phtopthora capsica sporangia. Phtopath 99: 1258-64.
8. Bowers JH, Papavizas GC, Johnston SA (1990) Effect of soil temperature and soil-water matric potential on the survival of Phytopthora capsici in natural soil. Plant Dis 74: 771-77.
9. Ristaino JB, Johnston SA (1999) Ecologically based approaches to management of Phytophthora blight on bell pepper. Plant Dis 83: 1080-89.
10. Granke LL, Ocampo LQ, Lamour K, Hausbeck MK (2012) Advances in research on Phytopthora capsici on vegetable crops in the United States. Plant Dis 11: 1588-1600.
11. Shilpa, Bijalwan P, Shukla YR (2023) Efficacy of different agro-techniques on growth, yield and disease incidence on tomato (Solanum lycopersicum L.) crop of north-western Himalayan region. Environ Con J doi.org/10.36953/ECJ.11152279.
12. Babadoost M (2004) Phytophthora blight: a serious threat to cucurbit industries. APSnet.doi:10.1094/APSnetFeature-2004-0404 Online.
13. Ram H, Singh Y, Timsina J, Humphreys E, Dhillon SS, Kumar K, Kler DS (2005) Performance of upland crops on raised beds in north western India. Evaluation and performance of permanent raised bed cropping systems in Asia, Australia and Mexico. ACIAR Proceedings No. 121. pp. 41-58.
14. Bijalwan P, Shilpa, Kaushik P (2022) Response of bell pepper (Capsicum annuum) to different agro-techniques and NAA application. Ind J Agric Sci 92: 1353-1357.
15. Bijalwan P, Shilpa, Shukla YR, Thakur KS, Gupta M (2022) Weed Management Efficacy of Mulches, Planting Methods and NAA Application in Capsicum annuum L. Under Mid-Hills of Himachal Pradesh. Ind J Agric Res DOI: 10.18805/IJARe. A-5993.
16. Kousik CS, Adams ML, Jester WR, Hassell R, Harrison HF, Holmes GJ (2011) Effect of cultural practices and fungicides on Phytophthora fruit rot of watermelon in the Carolinas. Crop Prod 30: 888-94.
17. Lightfoot DR (1994) Morphology and ecology of lithic-mulch agriculture. Geo Rev 25:172 85.
18. Ahmad S, Raza MAS, Saleem MF, Zahra SS, Khan IH, Ali M, Shahid AM, Iqbal R, Zaheer MS (2015) Mulching strategies for weeds control and water conservation in cotton. J Agric Bio Sci 8:299–306
19. Kader MA, Singha A, Begum MA, Jewel A, Khan FH, Khan NI (2019) Mulching as water-saving technique in dry land agriculture. Bull of the Nat Res Centre 43:1–6.
20. Iqbal R, Muhammad ASR, Muhammad FS, Imran HK, Salman A, Muhammad SZ, Muhammad U, Imran H (2019) Physiological and biochemical appraisal for mulching and partial rhizosphere drying of cotton. J Arid Land 11:785–94
21. Shilpa, Bijalwan P, Shukla YR, Kansal SK, Thakur KD (2022) Effect of crop management practices on growth, yield, quality, economics and buckeye rot, Alternaria disease of tomato (Solanum lycopersicum L.) var. Solan Lalima. Int J Environ & Climate Change 12: 1-14.
22. Ahmed M, Baiyeri KP, Echezona BC (2013) Effect of coloured polyethylene mulch and harvesting stage on growth and yield of industrial sugarcane in Nigeria. Afric J Biotech12:10–18
23. Kwambe XM, Masarirambi MT, Wahome PK, Oseni TO (2015) The effects of organic and inorganic mulches on growth and yield of green bean (Phaseolus vulgaris L.) in a semi-arid environment. Agric Biol J Am 6:81–89
24. Iqbal R, Raza MAS, Nazar MA (2020) Potential agricultural and environmental benefits of mulches-a review. Bull of the Nat Res Centre 44: 75. https://doi.org/10.1186/s42269-020-00290-3.
25. Chalker-Scott L (2007) Impact of mulches on landscape plants and the environment – a review. J Environ Hortic 25:239–249
26. Basuchaudhari P (2016) 1-Napthaleneacetic acid in rice cultivation. Curr Sci 110: 1.
27. Danesh TS, Mehrafarin A, Naghdi BH, Khalighi SF (2014) Changes in growth and trigonelline/mucilage production of fenugreek (Trigonellafoenum-graecum L.) under plant growth regulators application. J Med Plants 13: 15-25.
28. Kumari A, Singh VK, Shree S, Kumar V, Nirgude V (2017) Influence of naphthalene acetic acid (NAA) and integrated nutrient management (INM) on yield and economics attributes of chiili (Capsicum annuum L.). J of Appl and Nat Sci 9: 311-15.
29. Al-Masri MI, Barakat R (2003) Role of auxin, Napthalene Acetic Acid (NAA) in the pathogenicity of Sclerotinia sclerotiorum, the causative organism of white mold disease. Hebron Uni Res J 1, 2.
30. Elnora AS, Wightman F (1978) Auxins. In: Phytohormones and related compounds, ed. By Letham G and Higgins. Elsevier, North Holland Biomedical Press.
31. Quesasa-Ocampo LM, Fulbright DW, Hausbeck MK (2009) Susceptibility of fraser fir to Phytopthora capsica. Plant Dis 93: 135-41.
32. Sharma D, Kumar S, Rana KS (2016) Integrated management of collar rot of bell pepper. Int J of Agric Sci 8: 3468-70.
33. Locher J, Ombodi A, Kassai T, Dimeny J (2003) Effects of black plastic mulch and raised bed on soil temperature and yield of sweet pepper. Int J of Hortic Sci 9: 107-10.
34. Singh VK, Dwivedi BS, Shukla AK, Chauhan YS, Yadav RL (2005) Diversification of rice with pigeon pea in a rice-wheat cropping system on a typicustochrept: Effect on soil fertility, yield and nutrient use efficiency. Field Crops Res 92: 85-105.
35. Das A, Patel DP, Ramkrushna GI, Munda GC, Ngachan SV, Kumar M, Buragohain, Naropongla (2014) Crop diversification, crop and energy productivity under raised and sunken beds: results from a seven-year study in a high rainfall organic production system. Biological Agri and Horti 30: 73-87.
36. Brown JE, Stevens C, Osborn MC, Bryce HM (1989) Black plastic mulch and
    spun bonded polyester row cover as method of southern blight control in bell
    pepper. Plant Dis 73: 931-32.
37. Ashrafuzzaman M, Halim MA, Ismail MZ, Shahidullah SM, Hossain A (2011) Effect of plastic mulch on growth and yield of Chilli (Capsicum annuum L). Brazilian Archives of Biol and Tech 54: 321-30.
38. Verma R, Mehta DK, Thakur KS, Prasad H, Lal M (2016) Effect of mulching geometry on seed production of bell pepper (Capsicum annuum L.) in mid-hills of Himachal Pradesh. Int J Bio-resources and Stress Mgt 7: 1146-51.
39. Dhaliwal MS, Sharma SP, Jindal SK, Dhaliwal LK, Gaikwad AK (2017) Growth and yield of bell pepper as influenced by growing environment, mulch and planting. date J of Crop Improv 31: 830-46.
40. Edgar ON, Gweyi-Onyango JP, Korir NK (2016) Influence of Mulching Materials on the Growth and Yield Components of Green Pepper at Busia County in Kenya. Asian Res J of Agri 2: 1-10.
41. Nakamura T, Kawanabe Y, Takiyama E, Takahashi N, Murayama T (1978) Effects of auxin and gibberellin on conidial germination in Neurospora crassa. Plant Cell Physiol 19: 705-709.
42. Tomita K, Murayama T, Nakamura T (1984) Effects of auxin and gibberellin on elongation of young hyphae in Neurospora crassa. Plant Cell Physiol 25: 355-358.
43. Michnievicz M, Rozej B (1988) Is the gibberellin limiting factor for the growth and development of Fusarium culmorum? Acta Physiologiae Plantarum 10: 227-236.
44. Gare BN, Raundal PU, Burli AV (2017) Effect of plant growth regulators on growth, yield and yield attributing characters of rainfed chilli (Capsicum annuum L.). Adv Agric Res and Tech J 1: 195-97.
45. Fathima PS, Denesh GR (2014) Influence of humic acid spray on growth and yield of chilli (Capsicum annuum L.). Int J Agric Sci 9: 542-46.
46. Vandana PP, Varma LR (2014) Effect of spray treatment of plant growth substances at different stages on growth and yield of sweet pepper (Capsicum annuum L.) cv. Indra under green house. Int J Life Sci Res 2: 235-40.
47. French-Monar RD, Jones JB, Roberts PD (2006) “Characterization of natural populations of Phytophthora capsici associated with local weed populations in Florida vegetable farms”. Plant Dis 90: 345–50.
48. Keinath AP (2019) Aug. Managing Phytophthora capsici Diseases on Vegetables. Clemson (SC): Clemson Cooperative Extension, Land-Grant Press by Clemson Extension. LGP 1014.
49. Duniway JM (1983) Role of physical factors in the development of Phytophthora diseases. In: Erwin DC, Bartnicki-Garcia S and Tsao PH. ed. Phytophthora: its biology, taxonomy, ecology and pathology. St Paul, Minnesota, APS Press. p.175-87.
50. Vawdrey LL, Grice KE, Peterson RA (2004) Diversity and management of Phytopthora in South east Asia. ACIAR Monograph 114. pp 167-70.
51. Islam MS, Saif H, Islam MR, Naher Q, Khan ASMMR (2018) Soil salinity management for increasing potato yield in the coastal area of southern Bangladesh. Bangladesh J Agric Res 43: 655-68.
52. Belel MD (2012) Effects of grassed and synthetic mulching materials on growth and yield of sweet pepper (Capsicum annuum L.) in Mubi, Nigeria. J of Agric and Soci Sci 8: 97-99.
53. Bahadur A, Singh AK, Singh KP (2013) Effect of planting systems and mulching on soil hydrothermal regime, plant physiology, yield and water use efficiency in tomato. Ind J Hort 70: 48-53.
54. Mohammad MK, Rasdi MZMD, Naeimah NN, Nashriyah MAT (2017) Effects of naphthalene acetic acid (NAA) on the plant growth and sugars effects on the cut flowers Mokara CharkKuan orchid. Biosci J 33: 19-30.
55. Kumari A, Singh VK, Shree S, Kumar V, Nirgude V (2017) Influence of naphthalene acetic acid (NAA) and integrated nutrient management (INM) on yield and economics attributes of chiili (Capsicum annuum L.). J Appl & Natural Sci 9: 311-15.

Figure 1.  Interaction effect on severity of leaf blight (%) in bell pepper

Figure 2.  Interaction effect on incidence of collar rot (%) in bell pepper

Figure 3. Interaction effect on incidence of fruit rot (%) in bell pepper

Figure 4. Interaction effect on yield/plot (kg) of bell pepper

Figure 5. Interaction effect on yield/ha (q) of bell pepper

Table 1. Detail of treatments used in the experiment

Table 2. Effect of planting methods, mulches and NAA levels on diseases and yield of bell pepper

*Figures in parenthesis represent square root transformation

P₁: Raised bed planting method, P₂: Flatbed planting method, M₁: Black polythene mulch, M₂: Silver/black polythene mulch, M₃: No mulch, N₁: NAA application @ 15ppm at 30 and 45 days after transplanting, N₂: No NAA application

Table 3. Interaction effect of planting methods, mulches and NAA levels on disease and yield of bell pepper

*Figures in parenthesis represent square root transformation

P₁: Raised bed planting method, P₂: Flatbed planting method, M₁: Black polythene mulch, M₂: Silver/black polythene mulch, M₃: No mulch, N₁: NAA application @ 15ppm at 30 and 45 days after transplanting, N₂: No NAA application