Humic Substances Improve Vegetable Seedling Quality and Post-Transplant Yield Performance under Stress Conditions
Round 1
Reviewer 1 Report
In response to Your request, I make the following additional remarks to improve the article:
- Please include a table that contains the explanation for abbreviations - I find the presentation of the commercial humic acid preparation too short and not elaborated enough. What can be the stability constants of complex compounds for elelments such as iron, zinc and copper ? The effect needs to be explained (concentration ? chemical connections ? mobility of elements ? change of pH ? - such data of the commercial medium (CM) should also be provided, as there are large differences in the elements themselves and the concentration of elements. An assessment of this issue can answer the question of why the HS has a positive effect (element uptake possibilities, increase in concentration etc. ... ) More chemical characteristics should be given. - I dispute if the article should be given such a general title in case of a commercial product. - Acknowleding the tremendous work the authors put into the article in terms of measuring parameters for four plant species, I have to note that the large amount of data made the text very condensed. Figures 2, 3, 4 and 5 each consist of 28-28 small figures. - In spite of this I find the conclusion too general, even though the conclusions apply to only one product. The results are very good for practical use but about what causes the real impact there is no information.Author Response
In response to Your request, I make the following additional remarks to improve the article:
- Please include a table that contains the explanation for abbreviations
Reply: we added the abbreviations as a list in Appendix Table 1.
- I find the presentation of the commercial humic acid preparation too short and not elaborated enough. What can be the stability constants of complex compounds for elements such as iron, zinc and copper? The effect needs to be explained (concentration? chemical connections? mobility of elements? change of pH? - such data of the commercial medium (CM) should also be provided, as there are large differences in the elements themselves and the concentration of elements. An assessment of this issue can answer the question of why the HS has a positive effect (element uptake possibilities, increase in concentration etc. ... ) More chemical characteristics should be given.
Reply: thank you for pointing out this important aspect, this gives us more thinking about what’s the real “cause” for HS beneficial effects. We added a whole new paragraph (now the second paragraph in discussion - please see below). This paragraph explained more about this solid HS product, including concerns for the “cause” of the positive effects of HS. In general, we thought the element or nutrient differences were not the main reasons since we only applied HS with 1% v/v; the nutrient differences were minimized (please see following explanation and table 2). Instead, we thought the main reason is the root-promoting effects due to HS’s hormone-like effects. Still, it will be nice to measure the nutrient composition of transplants, and try to see if HS can improve their nutrient (element) uptake due to the improvement of root growth, but this study is mainly focused on transplant quality and subsequent yield. A future investigation for nutrient uptake would be interested to extend this work and contribute to further explanations for the positive effects.
Paragraph added “The HS used in this study were obtained by using ammonoxidation procedure (lignite reacting with oxygen in aqueous ammonia) and resulted in a product with lower hydrophobicity (mainly caused by reduced aromatic compounds) and higher bioactivity than naturally slow-generated HS from lignite [26]. In addition, solid-HS contains humin, which has less hydrophilic carboxyl and hydroxyl groups but higher hydrophobic alkyl group and ash contents [18]. Raw materials also decide HS properties: lignite-derived HS are composed of highly oxidized sulfur-containing molecules, aromatic and aliphatic groups, which can give the products a higher hydrophobic protection than other raw materials (e.g., peat, compost, sludge, leonardite). This makes them more stable in terms of their existence (life-span) in the soil solutions, having slowly beneficial effects [27,28]. This HS product contained higher N, K, Mg and Na contents than commercial media, however, by adding HS with 1% v/v, the nutrient differences compared with control (solely commercial media) were minimized. The similar early growth performance (4 or 5 WAS) also indicated that there were no initial nutrient differences between control and HS-treated trays. During the seedling growth period, the fertilization amount applied for both control and HS trays were exactly the same and sufficient for seedling growth, thus the beneficial effects from HS were probably not related with nutrients. We found the increased seedling biomass in HS-treated trays mainly occurred at a later seedling growth stage (6 WAS) and during early field establishment, with prominent effects on root development. This could indicate the positive results from HS were mainly due to their bio-stimulation (auxin-like) effects on enhancing plant root development and nutrient acquisition [13], which occurred slowly due to the solid-HS product. Since transplant quality was the main focus, below we explain in detail the effects of HS on the specific transplant growth parameters”
- I dispute if the article should be given such a general title in case of a commercial product.
Reply: we feel this HS product could be a representative solid HS product since it has humic acid as bio-active component and humin as a slowly decomposition component with longer existence in soil solutions. Few studies were conducted using HS (mostly liquid form) for vegetable transplant production, so we think the general title will raise awareness of this new area of research.
- Acknowledging the tremendous work, the authors put into the article in terms of measuring parameters for four plant species, I have to note that the large amount of data made the text very condensed. Figures 2, 3, 4 and 5 each consist of 28-28 small figures.
Reply: we thought that adding detailed results, this paper could provide more useful and robust information about the use of solid HS in vegetable transplant production since no one have used this kind of solid HS in similar studies. For example, by proving leaf, stem, and root growth data, we can find plant growth component has prominent responses due to HS application, and how their growth rate change dynamically. Detailed root to shoot ratio could also provide information about what’s the suitable R:S ratio for a good quality transplant. We tried to make the narrative of results simple, direct and not overly complex, and separated each cultivar responses with one paragraph.
- In spite of this I find the conclusion too general, even though the conclusions apply to only one product. The results are very good for practical use but about what causes the real impact there is no information.
Reply: thank you. We have revised the conclusion (see below) with detailed description for each crop. We think this product is a representative solid HS product as mentioned above, the “cause” of this positive effects was also explained and expanded in the discussion (please see reply above).
Revised: “In this study, humic substances (HS) added as a media amendment for growing containerized vegetable transplants, were evaluated for their seedling root and shoot growth modulation effects before and after field transplanting. Compared with control, HS: 1) improved plant shoot biomass accumulation of pepper, tomato and lettuce mostly due to faster shoot growth rates, while these effects were not prominent in watermelon; 2) enhanced pepper and watermelon root developmental traits (RDW, RL, RSA) after transplanting due to faster root growth rates, and tomato root development both before and after transplanting, while these effects were not shown in lettuce; 3) decreased net assimilation rate of tomato, watermelon and lettuce before transplanting but improved after transplanting, while this effect was not significant for pepper; 4) improved leaf area ratio in all four crops; 5) improved specific root length of tomato, watermelon and lettuce before transplanting but decreased it after transplanting; 6) lowered root to shoot ratio of all the crops before transplanting but reversed it after transplanting, except for lettuce. Based on the field performance, we found suitable R:S ranges for high quality transplants to be as follow: 0.25-0.35 for pepper, 0.15-0.2 for tomato, 0.1 for watermelon, and 0.15-0.2 for lettuce. This study demonstrated that HS differentially modulated root and shoot growth based on crop species: root performances were outstanding in fruit-based crops (pepper, tomato, watermelon), while leaf performances were significantly improved on the leaf-based crop (lettuce). Overall, imposed heat and drought stresses had significantly negative effects on crop yield and average fruit weight, but HS-treated plants showed improved stress tolerance than control plants by mitigating the yield loss. This study showed the potential application of solid humic substances as bio-stimulants for enhancing transplant quality and crop performance in four economically important vegetable species (tomato, pepper, watermelon and lettuce)”.
Reviewer 2 Report
The manuscript is interesting, suits the journal’s aim and scope well, but needs for minor correction.
The comments are below by the lines of the manuscript.
Lines 13, 14
HS were applied to a peat-based growth 13 medium (1% v/v) to evaluate…
This seems to be too high dose.
Lines 22-22
The negative effects of heat and drought stresses on crop yield were more prominent in control plants, while HS-treated transplants were able to mitigate yield decreases.
It would be better to prevent these effects in prior than to fight consequences. See and apply our Biogeosystem Technique methodology.
Line 42
…root to shoot balance in the confined cells – high transplant quality…
What is a criterion of this delicate balance?
Line 50
…nitrogen fertilization rate…
What about conditions to atmospheric nitrogen fixation by soil biota?
Line 50
… irrigation systems…
It is well known fact that current irrigation spoils too much water and leads to soil degradation. Have you any propositions for containerized transplant improved water regime?
Line 81, 82
…peat-based growing media…
What properties growing media has?
Line 108
Table 1. Basic physical properties of the commercial media (CM) and humic substances (HS).
Line 108
CM bulk density 0.07
Do you insist on this value? Is not it too low?
Line 114
Please characterize the irrigation system in some details.
Line 117
Please some details about conditions which where provided by Wadsworth control system.
Line 121
Please give a soil name.
Line 122
What were tillage and other soil conditions for the future agrobioceonosis?
Line 125
This is subsoil drip irrigation. Please explain the irrigation in some details.
Line 132
How can you divide a heat stress and a drought stress in the field experiment?
Line 134
And what was a reduction of water supply volume to the field square unit?
Line 264
You mean high soluble salt content as a property of any compost?
If so, irrigation is capable to correct the disadvantage.
Line 272
What was an improvement degree criterion?
Line 292
ratio?
Line 343
It was not a good idea to finalize your own research, basing on the other researchers' opinion. Maybe the Figure 7 is your own?
Please give clear explanations, basing on your own data.
Line 362
You have much data in your text, tables and figures to show the individuality of your conclusions.
Author Response
The manuscript is interesting, suits the journal’s aim and scope well, but needs for minor correction. The comments are below by the lines of the manuscript.
Lines 13, 14
HS were applied to a peat-based growth medium (1% v/v) to evaluate…This seems to be too high dose.
Reply: the humic substances we used is a solid product, which will have a longer existence in the solution and relatively slower effects than liquid HS product (such as extractant humic acids). We used 1% HS as volume based on previous seed germination tests which showed 1% v/v didn’t have phytotoxic effects (actually 5% had phytotoxic effects based on our preliminary test).
Lines 22-22
The negative effects of heat and drought stresses on crop yield were more prominent in control plants, while HS-treated transplants were able to mitigate yield decreases.
It would be better to prevent these effects in prior than to fight consequences. See and apply our Biogeosystem Technique methodology.
Reply: we intentionally grew transplants under heat stress (during summer season) and drought stress (applied less irrigation) conditions. We wanted to test if HS-treated transplant with improved quality can mitigate these stresses that are causing yield decrease in control untreated plants. We agree, it will always be important to prevent transplanting of seedlings under unsuitable conditions. Biogeosystem Technique is a good method in sustainable agriculture for improving plant immunity and soil quality (as mentioned by Kalinitchenko V.P. et al., 2019, Biogeosystem technique methods for increasing immunity of plants). We can use this concept in future field-related studies, but this current study is mainly focused on transplant quality related parameters, so might not fit well.
Line 42
…root to shoot balance in the confined cells – high transplant quality…
What is a criterion of this delicate balance?
Reply: the balance in general can be reflected by the root:shoot (R:S) ratio, which is highly variable depending on crops and cultivars. For example, a good quality lettuce seedling have a R:S ratio ranging from 0.07-0.15 (Kerbiriou P. J. et al., 2013), but cultivars have different “best” ratios, some below 0.1, some above 0.13. Based on our results, the criterion can be defined as HS-treated results, as for pepper better R:S ratio ranging from 0.25-0.35, for tomato a R:S ratio ranging from 0.15-0.2, for watermelon a R:S ratio around 0.1, for lettuce better a R:S ratio ranging from 0.15-0.2. We added this information in conclusions as indicated by “Track Changes”.
Line 50
…nitrogen fertilization rate…
What about conditions to atmospheric nitrogen fixation by soil biota?
Reply: in transplant growth system, the biota is really limited in growth media (mostly peat or bark-based substrates) unless we introduce soil biota to growth media. Although it’s not related to our study, we found an interesting study from (Thangavelu Muthukumar, et al., Growth response and nutrient utilization of Casuarina equisetifolia seedlings inoculated with bioinoculants under tropical nursery conditions), they inoculated plant growth promoting biota (for benefiting nutrient uptake), and found that seedling quality was improved, although “microbial inoculation generally reduced the efficiency of nutrient utilization in dry matter production (nutrient use efficiency)”. Therefore, we think by introducing nitrogen fixation soil biota in growth media, the transplant quality could be increased, and less N is required.
Line 50
… irrigation systems…
It is well known fact that current irrigation spoils too much water and leads to soil degradation. Have you any propositions for containerized transplant improved water regime?
Reply: that’s a good point, in transplant production, which normally use overhead spray irrigation system, water use is relatively efficient than other methods applied to soils. But if we have a stronger transplant (which showed in our results) that good quality transplant can prevent yield reduction caused by drought stress; this might provide evidence that by producing a high-quality transplant, we might be able to reduce (slightly) water use in field conditions.
Line 81, 82
…peat-based growing media…
What properties growing media has?
Line 108
Table 1. Basic physical properties of the commercial media (CM) and humic substances (HS).
Reply: yes, we showed properties in table 1. This commercial media has 90% sphagnum peat.
Line 108
CM bulk density 0.07
Do you insist on this value? Is not it too low?
Reply: the value is correct, because the sphagnum moss-peat percentage is very high (90%) in the media (which normally has density ranging from 0.06-0.10 g/cm3, data from Fonteno, W. et al., 1995. Procedures for Determining Physical Properties of Horticultural Substrates Using the NCSU Porometer), so this media is very light.
Line 114
Please characterize the irrigation system in some details.
Reply: added details as “total length 7.1 m with two long arms at side and operating orbit at center, each arm has 3.2 m length with 13 sprinkler units”.
Line 117
Please some details about conditions which where provided by Wadsworth control system.
Reply: added details with “temperature and humidity” after Environmental conditions and “hourly” before monitored by a weather station.
Line 121
Please give a soil name.
Reply: the type of the soil is clay soil (not clay loam or silty clay) based on their composition of clay, sandy and silt.
Line 122
What were tillage and other soil conditions for the future agrobioceonosis?
Reply: added details with “Field was prepared using ridge tillage”. The field was covered with plastic mulch (except lettuce) after tillage, the initial soil characteristics are shown in table 2.
Line 125
This is subsoil drip irrigation. Please explain the irrigation in some details.
Reply: added details with “Drip irrigation with emitter rate at 0.87 L per hour and emitter spacing at 30 cm (Netafim, Fresno, CA, USA) was installed”.
Line 132
How can you divide a heat stress and a drought stress in the field experiment?
Reply: heat stress was applied by planting in 2 different seasons (the cool season started in early spring, the hot season started in late spring and early summer). In each season (considered cool or heat treatment), drought was applied for half of the plots along with no-drought for the other half plots. So, in the cool season, we have drought and no-drought treatments; in the hot season, we also have drought and no-drought treatments. Plants grown in hot season and under drought will have both stresses, and in no-drought treatment, plants will have sufficient irrigation based on ET so they will only have heat stress.
Line 134
And what was a reduction of water supply volume to the field square unit?
Reply: in general, 50% ET will have around 35%-40% less water applied than 100% ET per square unit (unit for irrigation amount is “mm”).
Line 264
You mean high soluble salt content as a property of any compost?
If so, irrigation is capable to correct the disadvantage.
Reply: composts that derived from organic water such as food waste, manure have high soluble salt content. We added this detail. Irrigation could mitigate this disadvantage, but too much water will also damage seed germination.
Line 272
What was an improvement degree criterion?
Reply: as shown in Appendix Table 3, we set up the significant level at P < 0.1, when they showed difference at this level, we know the improvement or reduction is worth to notice.
Line 292
ratio?
Reply: yes, we corrected this, thanks for pointing out.
Line 343
It was not a good idea to finalize your own research, basing on the other researchers' opinion. Maybe the Figure 7 is your own?
Please give clear explanations, basing on your own data.
Reply: we created Figure 7 for better understanding of the general effects of HS on all the 4 crops (each with 2 cultivars) we tested. In this figure, we found post yield performance is highly correlated with plant root development (biomass, surface area, root length), so we pointed out that other management strategies that can improve root growth are highly suggested in order to improve crop yield. We added a sentence at the end to make it clearer as “Overall, solid HS with shoot and root growth promoting effects can satisfy the requirements of transplant growth and subsequent yield in both pre- and post-transplanting environments, which makes them suitable and reliable amendments for use in transplant media”.
Line 362
You have much data in your text, tables and figures to show the individuality of your conclusions.
Reply: we added more details in the conclusions as “In this study, humic substances (HS) added as a media amendment for growing containerized vegetable transplants, were evaluated for their seedling root and shoot growth modulation effects before and after field transplanting. Compared with control, HS: 1) improved plant shoot biomass accumulation of pepper, tomato and lettuce mostly due to faster shoot growth rates, while these effects were not prominent in watermelon; 2) enhanced pepper and watermelon root developmental traits (RDW, RL, RSA) after transplanting due to faster root growth rates, and tomato root development both before and after transplanting, while these effects were not shown in lettuce; 3) decreased net assimilation rate of tomato, watermelon and lettuce before transplanting but improved after transplanting, while this effect was not significant for pepper; 4) improved leaf area ratio in all four crops; 5) improved specific root length of tomato, watermelon and lettuce before transplanting but decreased it after transplanting; 6) lowered root to shoot ratio of all the crops before transplanting but reversed it after transplanting, except for lettuce. Based on the field performance, we found suitable R:S ranges for high quality transplants to be as follow: 0.25-0.35 for pepper, 0.15-0.2 for tomato, 0.1 for watermelon, and 0.15-0.2 for lettuce. This study demonstrated that HS differentially modulated root and shoot growth based on crop species: root performances were outstanding in fruit-based crops (pepper, tomato, watermelon), while leaf performances were significantly improved on the leaf-based crop (lettuce). Overall, imposed heat and drought stresses had significantly negative effects on crop yield and average fruit weight, but HS-treated plants showed improved stress tolerance than control plants by mitigating the yield loss. This study showed the potential application of solid humic substances as bio-stimulants for enhancing transplant quality and crop performance in four economically important vegetable species (tomato, pepper, watermelon and lettuce)”.
