text:
Quality of Eucheuma cottonii seaweed cultivated in Lampung waters

This study was aimed to observe the quality of Eucheuma cottonii dried seaweed produced from seaweed farmers in Lampung, especially in its main production centers namely sub-district Ketapang, south Lampung. Seaweed was taken from three locations namely Ruguk, Legundi and Tri Dharma Yoga Village. The parameters observed were included seaweed moisture content, clean anhydrous weed (CAW), ash content, yield, and the quality of carrageenan produced. The results showed that seaweed moisture content ranged from 21.71 to 33.48%, CAW 37.53-40.72%, ash content 33.68-43.08%, and carrageenan yield 11.35-13.57%. From the results of the observation it was concluded that the quality of seaweed from Legundi Village was the best compared to other villages.

Introduction
E. cottonii is a seaweed producing carrageenan that is commonly used as raw material for carrageenan industries. Carrageenan used as additives for the food, beverage, cosmetics, pharmaceutical, paint, textile, and other industries. Besides being used as a food source, E. cottonii is also used as a producer of growth hormone that is often used for organic fertilizers (Sedayu et al 2013). Therefore the demand for this type of seaweed increases by 5-10% every year both to meet the needs of the seaweed industry in the country and for export. At present, Indonesia is the largest producer of E. cottonii seaweed in the world. This is supported by the development of seaweed cultivation technology that can be done with various techniques (Failu et al 2016, Soenardjo 211).
The price of seaweed on the market is very determined by the quality of seaweed. The quality of dried seaweed is regulated in Indonesian National Standards or Standar Nasional Indonesia (SNI). Seaweed must meet the standards issued by SNI 2690-2015, especially for the type of Eucheuma sp. seaweed has moisture content must be less than 30%, and clean anhydrous weed (CAW) minimum is 50%. To get good quality seaweed, seaweed handling techniques must be considered since harvesting, drying, packaging, and storage. Lampung is one of the main producers of seaweed E. cottonii in Sumatra, and most of this seaweed was produced by sub-districts of Ketapang. This area is the main producer of E. cottonii seaweed in Sumatra which is used as a source of seeds and as a source of raw material for the seaweed processing industry. The three main E. cottonii producers in Ketapang are Ruguk, Legundi and Tri Dharma Yoga villages. Different location characteristics between the three locations are thought to affect the quality of seaweed produced. Therefore this study aims to determine the quality of E. cottonii seaweed produced from Lampung, especially Ketapang sub-district as the main producer area, and further looking at the differences in the quality of seaweed between the three villages.

Materials
Seaweed samples were taken from three locations namely Ruguk, Legundi and Tri Dharma Yoga Villages at Ketapang sub-districts. dried seaweeds were collected from seaweed farmers, then packaged in plastic bags and closed tightly to be taken to chemical and processing products laboratories at BBRP2BKP Jakarta to observe the quality.

Methods
The parameters observed were included seaweed moisture content, clean anhydrous weed (CAW), ash content, yield, and the quality of carrageenan produced. During the research, interviews were also conducted with farmers about how to handle post-harvest and sale of seaweed products. Analysis of moisture and ash content were carried out using gravimetric method according to the method SNI 01-2354.2-2006. Clean anhydrous weed was analyzed using gravimetric method according to the method SNI 8168-2015. Carrageenan yields are analyzed according to the procedure SNI 2354.12-2013. To see the significant difference in the quality of seaweed between the three locations, analysis of variant (ANOVA) test was conducted and continued with the least significant difference (LSD) test with the SPSS program.

Results and discussion
From observations in the field, it appears that post-harvest handling of E. cottonii seaweed at the farmer level includes harvesting from the sea, washing with seawater, releasing from straps, cleaning of material and other grasses that attach, drying and packaging. The quality of dried seaweed from all three locations varies, this is related to sources and post-harvest methods at the farm level. The way farmers deal with seaweed has a huge influence on the quality of the produced seaweed.

Moisture content
The moisture content of seaweed is a parameter that shows the level of seaweed dryness and is closely related to the quality of seaweed during storage and distribution Moisture content of dried seaweed from three locations were varied from 21.71+0.44 to 33.48+0.73% (figure 1). Statistical tests showed that the seaweed of the three locations had significantly different moisture content (P<0.05). The highest moisture content found in seaweed from Tri Dharma Yoga Village, while the lowest is obtained from Legundi Village. If compared to the Indonesian standard quality for dry seaweed which requires a maximum moisture content of 30% (SNI 2690, it can be seen that the quality of seaweed from Legundi and Ruguk villages has met the requirements, while the origin of Tri Dharma Yoga still does not meet. The moisture content of seaweed is very closely related to the drying technique carried out by farmers. The high water content indicates that the drying process is still not optimal or the drying time is lacking. Some farmers tend to dry the seaweed in a short time to reduce the loss of seaweed weights produced. This will actually have an impact on the low price of seaweed because traders will assess the low  Tamaheang et al (2017) report that if drying is done correctly, with sunlight for 24 hours it is possible to produce seaweed moisture content 13.75%.

Ash content
Seaweed ash content from Lampung varies from 33.68±1.38 to 43.08±2.43% (table 1). The results of statistical tests show that there were significant differences between seaweed from Ruguk and other locations (P< 0.05). The high level of seaweed ash is not beneficial because it can cause high levels of carrageenan ash produced. Another disadvantage of the high ash content of seaweed is the need for an extra washing effort to clean seaweed before the carrageenan extraction process. Research Harun et al (2013) showed that the levels of carrageenan ash from the waters of the Tihenggo Village, Gorontalo City range from 14.62 to 18.41%. Indonesia's national standards for the quality of carrageenan require ash content between 15-40% (SNI 8391-1-2017).
From this study, it was seen that the ash content of dried seaweed from Lampung was still quite high. The high ash content indicates that there are still high levels of inorganic impurities such as sand or the high mineral content in seaweed. This is partly due to washing using seawater that is not clean enough and there is soil or sand contamination during the drying process.

Clean anhydrous weed
Clean anhydrous weed (CAW) is a parameter that shows the level of cleanliness of dried seaweed. The higher the CAW value, the cleaner and drier the seaweed will be, which will have an impact on the higher total dry weight available and carrageenan yield. CAW is one of the parameters required in the Indonesian national standard (SNI) for the quality and safety of dried seaweed products, with a minimum value of 50%.
The results of the study showed that clean anhydrous weed (CAW) seaweed from all three locations ranged from 37.53±1.15 to 40.72±4.23% ( figure 2) of dried seaweed from the three locations still does not meet the SNI quality requirements. The low value of CAW is caused by poor post-harvest handling at the farm level such as nonoptimal process of washing, cleaning, and drying of seaweed. This is also evident from the high ash and moisture content of seaweed ( figure 1 and table 1).

Carrageenan yield
Carrageenan yield is a quite important parameter because it determines the price of seaweed used as raw material for the carrageenan industry. The yield of carrageenan is very much determined by the quality of dried seaweed, and is influenced by the quality of planted seaweed used, harvest age, location and method of cultivation, depth of plantation, planting season, water quality, and post-harvest handling of seaweed and (Asni 2015, Failu et al 2016, Harun et al 2013, Kushartono et al 2009, Nur et al 2016and Soenardjo 2011. The results showed that carrageenan yield of these seaweed were ranging between 11.35+0.35 to 13.58+0.39%. There was a statistically significant difference in the yield of carrageenan from seaweed from the three locations, where the yield of carrageenan from legundi village was lower than that of other villages (P<0.05). In general, the yield of carrageenan from the three villages seaweed is still low compared to the results of other studies. Ega et al (2016), reported that carrageenan yield of E. cottonii seaweed extracted with different alkaline treatments ranging from 34.43 to 45.26%. Failu et al (2016) reported that yield of carrageenan from Kappaphycus alvarezii seaweed that cultivated using net-basket method was 46.76%. Harun et al (2013) reported the yield of carrageenan from Kappaphycus alvarezii seaweed with a different harvest age was varied from 23.01-30.63%. The low carrageenan yield obtained from seaweed from these villages is due to the still less optimal process of cleaning and drying seaweed. The high water content and impurities are seen from the low CAW value cause the low yield produced. The low quality of seaweed causes low prices received by

Marketing chain of seaweed and quality improvement by seaweed traders/distributors
From interviews with seaweed farmers and traders, seaweed marketing in Lampung is generally through the chain of farmers, small traders, big traders or distributors and industry or exporters ( figure 3). The quality of seaweed produced by farmers sometimes does not meet the quality requirements for the carrageenan industry or seaweed exporter, so the role of big traders/distributors is very important to improve the quality of seaweed. Seaweed quality improvement is generally carried out at big traders/distributor before being sent to industry or exporters. At the level of small traders, generally there is no improvement in the quality of seaweed that occurs. Small traders only collect seaweed from farmers, temporarily store it and send it to wholesalers.
The low quality of seaweed at the farm level is partly due to the farmers' lack of understanding of the quality of dried seaweed. In addition, some aquaculture conditions such as high turbidity of seawater and lack of washing processes also cause large amounts of impurities when drying seaweed. Another factor that causes low quality is a short drying process because farmers want to quickly sell their products to get money.
Improving the quality of seaweed at the level of wholesalers/distributors is generally done by cleaning dried seaweed from impurities that stick to it such as sand, soil, salt, and other impurities. Then the seaweed is dried again for several hours until the water content meets the industry/exporter requirements. seaweed is then packaged in sack packs, pressed and stored in the warehouse to wait for delivery to the industry or exporter. In conclusion, seaweed Legundi Village was high quality compared to other village.


provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2020.jsonl.gz.gz

text:
Efficacy of Organic Amendments and a Nematicide for the Management of Root-Knot Nematode (Meloidogyne incognita) of Tomato

A field experiment was conducted in the field of Plant Pathology Division, Bangladesh Agricultural Research Institute to find out the efficacy of organic amendments viz. poultry refuse, rice bran , fresh saw dust, dry saw dust, tea waste and a nematicide Furadan 5G against root-knot disease (Meloidogyne incognita) of tomato during three consecutive years viz. 2012-13, 2013-14 and 2014-15. Soil was treated with different organic amendments 10 days before seedling transplanting and Furadan 5G was applied on the day of seedling transplanting of tomato. The soils of the experimental plots were inoculated with chopped severely galled (M. incognita) roots of tomato at the time of treatment application. In all the years, considerable reduction in root-knot disease and increase in plant growth and fruit yield were achieved with different treatments and Furadan 5G. The most effective treatment was poultry refuse followed by rice bran and Furadan 5G. In 1 year, 2 year and 3 year, gall index values were 5.16, 4.94 and 6.27 under control, respectively. The severity was reduced to 59.11-64.53% in 1 year, 50.60-60.72% in 2 year and 48.96-55.34% in the 3 year compared to control due to application of the rice bran, Furadan 5G and poultry refuse. On the other hand, fruit yield under control was 44.82 t/ha at 1 year 49.73 t/ha in 2 year and 36.95 t/ha at 3 year. The highly effective three treatments increased fruit yield to 11.02-29.90% in 1 year, 17.17-30.56% in 2 year and 23.39-35.02% in the 3 year compared to control. The fruit yield of tomato was directly and linearly correlated with gall indices in tomato gall. Based on the findings of present the study poultry refuse and rice bran noted as an effective treatment to manage root-knot disease of tomato.

Introduction
Tomato (Lycopersicon esculentum L.) is one of the most popular and nutritious vegetable crops in Bangladesh which belongs to the family Solanaceae. It ranks next to potato and sweet potato in respect of vegetable production in the world (1,2) and tops the list of conned vegetables (3). The average yield of tomato in Bangladesh is 14.05 t/ha (4) which is quite low as compared to that of other tomato producing countries in the World (5). The low yield was mainly attributed to the susceptible nature of the crop to insect pests and diseases among which plant-parasitic nematodes are one of the most prevalent (6). Plant-parasitic nematodes have been implicated as a major constraint to tomato production (7). In general, the most widespread nematode species are the root-knot nematodes (Meloidogyne spp.). The root-knot nematodes are an economically important group of plant-parasitic nematodes (8). The common root-knot nematodes identified as parasites of tomatoes in the tropics are: M. incognita (Kofoid and White), M. javanica (Treub), and M. arenaria (Neal) of which M. incognita is the most important (9). Yield loss of about 20.6% in tomatoes has been attributed to Meloidogyne species (10,11). It causes about 40% yield loss of tomato in Bangladesh and about 46.2% yield reduction in India (12,13). A number of approaches aimed to control root-knot nematodes through application of nematicides (14), organic soil amendments (15,16,17,18), cultural management, physical methods like soil solarization and biological measures like Trichoderma spp, Pacecilomyces lilacinus, Pasturia penetrans and Pseudomonas aeruginosa (19,20,21). Tomato cultivar resistant to root-knot nematode is not available in Bangladesh. For many years chemical nematicides have been used to control plant parasitic nematodes effectively. Although these are effective and fast acting, they are degrading to the environment, other beneficial soil micro flora and human health (22). Therefore, alternate management options against the nematodes are to be sought. Organic amendments derived from livestock manure, sewage wastes and different composts have been reported to have an effect on plant parasitic nematodes and free living micro flora (23, 24 25, 26). Organic amendments have been identified to enhance soil fertility and soil microbial populations (27), as well as suppress plant-parasitic nematodes (28). Therefore, the present study was undertaken to identify organic amendments with potentials to increase yield of tomato and to reduce root-knot nematode of tomato.

Materials and Methods
A field experiment was conducted to test efficacy of available organic amendments namely saw dust, rice bran , poultry refuses containing bedding materials and tea waste and a nematicide Furadan 5G (Carbofuran) to control root-knot nematode of tomato. The experiment was conducted in three consecutive years viz. 2012-13, 2013-14 and 2014-15 in the field of Plant Pathology Division BARI, Joydebpur, Gazipur. A total of 7 treatments including a control viz. i) Furadan (F) 5G @ 40 kg/ha, ii) Fresh saw dust @ 2.5 t/ha, iii) Dry saw dust @ 2.0 t/ha, iv) Rice bran @ 2.0 t/ha, v) Poultry refuse @ 5 t/ha, vi) Tea waste @ 2 t/ha and vii) Untreated control. The experiment was laid out in a randomized complete block design with 3 replications. The unit plot size 3m x 2.5m keeping 1m distance from plot to plot. Standard cultivation procedures recommended by BARI were followed to grow tomato with little modification. The experimental land was prepared with proper tillage and fertilizers were added during final land preparation. Requisite quantity of all organic amendments were incorporated with the soil 10 days before transplanting of tomato seedlings and allowed to decompose properly. Furadan 5G was applied in the field just before transplanting of seedlings. To ensure inocula of the nematode, chopped severely galled tomato roots infected with M. incognita were mixed with soil around the tomato seedlings @ 2 g/plant. Twenty five days old and apparently healthy tomato seedlings of variety BARI Tomato-2 (Ratan) were transplanted in the experimental plots maintaining row to row and plant to plant distance of 60 cm and 50 cm, respectively. Ten additional seedlings per plot were also transplanted in between two rows. During crop season necessary weeding, irrigation and other intercultural operations were done as per recommendation of the crop (29).

Data Collection
Data on shoot length, shoot weight, root length, root weight, gall index and yield were recorded. The root-knot disease severity was recorded two time one at 65 days after transplanting and another one after final harvest of tomato. The additional ten plants per plot were carefully uprooted after 65 days of transplanting and the root systems were cleaned with running tap water. Data on length and weight of shoot and root were recorded. The severity of root gall was recorded in terms of gall index based on a 0-10 scale (30). Data on fruit yield were recorded from five randomly selected plants per plot. The fruit yield was expressed in t/ha.  (Table 1). In the first year, the highest shoot length was obtained with poultry refuse followed by rice bran, tea waste, Furadan 5G and fresh saw dust. The lowest shoot length was recorded from control. More or less similar results also observed in the second year where the maximum shoot length was recorded from plots treated with poultry refuse by 75.55 cm/plant. In the third year maximum shoot length was recorded from the poultry refuse and rice bran treated plots followed by fresh saw dust and Furadan 5G. The least effective treatments to increase shoot length was dry saw dust followed by tea waste (Table 1).

Results
In the first year, the shoot weight of tomato was only 218.3 g/plant under control (Table 1). It increased to 231.7.0-347.2 g/plant due to treatments with organic amendments and Furadan 5G. The highest shoot weight was achieved with poultry refuse followed by rice bran, Furadan 5G and fresh saw dust. The least effective treatment to increase shoot weight was dry saw dust, which was followed by tea waste. In the second year, the shoot weight of tomato was 268.3 g/plant in control. Application of poultry refuse gave the highest shoot weight of 466.8 g/plant followed by rice bran , Furadan 5G and fresh saw dust where the shoot weight was 393.3 g/plant, 368.3 g/plant and 344.4 g/plant, respectively (Table 1). In the third year application different treatments gave higher shoot weight over control within the range of 154.70-236.50 g/plant. Two treatments with poultry refuse and rice bran yielded higher shoot weight. The least effective treatment to increase shoot weight was dry saw dust followed by tea waste (Table 1). Root growth: Amendment of soil with poultry refuse, rice bran and fresh saw dust and application of Furadan 5G showed positive effects on root growth of tomato as compared to control (  Table 2). The least effective treatment was dry saw dust followed by tea waste and fresh saw dust. In the third year, the minimum root weight was 8.43 g/plant in control. The maximum root weight was 15.80 g/plant in plot treated with poultry refuse followed by rice bran, Furadan 5 G, tea waste and fresh saw dust. Severity of root gall: In all the years, the severity of root gall of tomato was drastically reduced over control due to treatment of soil with organic amendments viz. poultry refuse, rice bran , saw dust and application of Furadan 5G (Table 3). In the first year, the maximum average gall index value of 5.16 was recorded in the control plot. It was reduced to 1.83 to 3.45 due to treatments with organic amendments and the Furadan 5G. The lowest severity of root-knot disease of tomato was recorded from the treatment with poultry refuse which was followed by Furadan 5G, rice bran , fresh saw dust and tea waste but all the treatments are statistically identical except dry saw dust. Application of poultry refuse gave 64.53% reduction of root knot disease severity compared to control followed by Furadan 5G, rice bran, fresh saw dust and tea waste where the reduction disease severity was 60.08%, 59.11%, 55.81% and 52.71%, respectively compared to control ( Table 3).
In the second year, the highest gall index value of 4.94 was found in control plot and the values were reduced to 1.94 to 3.11 due to application of different treatments ( Table 3). The reduction in disease severity was significant compared to control. The maximum reduction 60.72% over control was obtained with poultry refuse followed by Furadan 5G, rice bran and fresh saw dust with the reduction of 51.82%, 50.60% and 47.17% respectively compared to control. The least effective treatment to reduce root galling was dry saw dust followed by tea waste (Table 3). More or less similar trend also observed in the third year where the maximum gall index value of 6.27 was found in the control treatment. Application of poultry refuse gave the maximum 55.34% reduction of root knot disease severity compared to control followed by Furadan 5G and rice bran with 51.03% and 58.96% reduction of root knot disease severity than control.
Crop yield: Organic soil amendments and Furadan 5G gave appreciable increase in fruit number/plant and fruit yield per hectare in all the years (Table 4). However, yield increase was not significant under all treatments compared to control (Table 4).
Under control, fruit number per plant was 23.11 in the 1 st year, 23.60 in the 2 nd year and 20.53 in the 3 rd year (Table  4). Fruit number per plant increased to 23. 36-29.11 in the 1 st year , 26.27-32.27 in the 2 nd year and 25.07-32.40 in the 3 rd year due to different treatments. In the 1 st year, only the treatments with poultry refuse increased fruit number significantly over control. In the 2 nd year, all treatments with poultry refuse, rice bran and Furadan 5G caused significant increase in fruit number over control. The lowest increase 10.16% than control was achieved with dry saw dust followed by tea waste and fresh saw dust treatments with increased 10.61% and 10.84%, respectively compared to control. Poultry refuse gave the highest increased of fruit number with 26.87% over control, which statistically similar to rice bran (Table 4). In the 3 rd year, all the treatments significantly increased fruits number per plant compared to control. The highest fruits number per plant was 32.40 by poultry refuse treatment which is significantly differed from other treatments. Soil treatment with poultry refuse gave 36.64% higher yield than control which was followed by rice bran , fresh saw dust and Furadan 5G where the fruits number was 25.07%, 23.96% and 23.60%, respectively higher than control (Table 4).
In the first year, the lowest fruit yield of 44.82 t/ha was found under control ( Table 4). The yield was increased to 46.53 to 58.22 t/ha due to application of different treatments with poultry refuses, rice bran, Furadan 5 G and saw dust. The maximum yield was obtained with poultry refuse where fruit yield was 29.90% higher compared to control which followed by Furadan5G with 16.47% higher yield than control (Table 5). Differences in fruit yield harvested from control plots and plots treated with rice bran , saw dust and tea waste was not significant.   In the 2 nd year, average fruit yield was 49.73 t/ha under control and 54.56 to 71.62 t/ha under treated plots ( Table 4). The highest yield was 71.62 t/ha obtained from poultry refuse treatment followed by rice bran and Furadan 5G with the yield of 64.40 and 60.04 t/ha, respectively. The highest increase of yield 30.56% compared to control in poultry refuse treatment which was followed by rice bran Furadan 5G and fresh saw dust where yield was increase 22.77%, 17.17% and 15.81%, respectively over control. The lowest increase 8.85% than control was achieved with tea waste followed by dry saw dust.
In the 3 rd year, average fruit yield was 36.95 t/ha under control treatment and the yield was increase 45.35 to 56.86 t/ha under treated plots ( Table 4). The yield increase over control was significant under all treatments with poultry refuse, Furadan 5G and rice bran. The highest yield was obtained with poultry, which was statistically similar to Furadan 5G. Soil amendment with poultry refuse gave the highest increase of yield 35.02 compared to control which was followed by Furadan 5G and rice bran where the yield increase was 28.25% and 23.39%, respectively over control. The lowest yield increase 18.52% than control was found under dry saw dust followed by tea waste with 19.64% higher yield compared to control.
Correlation and regression analysis was performed to find out the relationship of fruit yield, shoot weight and root weight with gall index values of tomato grown in soil inoculated with M. incognita and treated with poultry refuse, rice bran , saw dust, tea waste and Furadan 5G ( Figure 1). Pooled data on those parameters recorded in three consecutive years were used for this analysis and found that the relationship was linear and negative for fruit yield, shoot weight and root weight with coefficient of correlations (r) 0.820, 0.673 and 0.755, respectively, The relationship was significant in case of fruit yield, shoot weight and root weight and influence of gall index on those parameters may be attributed to 67.30% (R 2 =0.673), 45.30% (R 2 =0.453) and 57.00% R 2 =0.570), respectively. The results indicated that organic amendments improved plant growth. It may be due to addition of plant nutrients to the soil. A Lower R 2 value indicates that other factors are also involved in plant growth and yield increase.

Discussion
Plant parasitic nematodes especially root knot nematodes are the major constraint to vegetables production all over the world. Organic amendments have been recognized in the management of plant parasitic nematodes and improvement of soil health. Due to their apparent environmental nontoxic benefits they have been considered in integrated nematode management with inorganic amendments. The present study was designed to determine the potential of organic amendment viz. poultry refuse, rice bran , fresh and dry saw dust and tea waste in the suppression of root-knot nematodes and increasing plant growth as well as yield of tomato in the field. Our results demonstrated that amending field soil with organic amendment viz. poultry refuse, rice bran , fresh and dry saw dust and tea waste suppressed gall index valued caused by root-knot nematode M. incognita and increasing plant growth parameters such as shoot length, shoot weight, root length and root weight as well as yield of tomato compared to control. These results are in agreement with that of McSorley and Gallager (31) and Kimpinski et al. (32) who reported that organic amendments from various sources reduce nematode populations. Results of the present study reveal that soil amendment with organic amendments effectively reduced root-knot severity and increased plant growth and fruit yield of tomato grown in M. incognita inoculated soil. Chen et al. (33) reported that use of organic amendments in the form of manure and compost effectively decrease parasitic nematode populations and disease intensity on plants. The nematicidal effects of organic amendments may be act directly or indirectly on plant parasitic nematodes. Applications of organic amendments change the soil physical and chemical properties which can improve plant health and making the plants to be more tolerant to nematode and other pathogenic attacks. Nematicidal effects of organic amendments have been attributed to several factors including increase in facultative parasites due to their richness in organic matter and release of toxic substances during decomposition (34,35). Oka (36) reported that the mechanisms of organic amendments for diseases suppression could be by enhancing the plant resistance through improving the physical, chemical and biological characteristics of the soil. Renco and Kovacik, (23) reported that organic amendments have the ability to not only stimulate beneficial and free living nematodes, but also other important micro flora around the plant rhizosphere that are antagonistic to parasitic nematodes. Decomposing plant residues and other organic amendments release compounds or by products such as nitrogen and organic acids that may have nematicidal effects (36,37). The results of the present study clearly indicated that poultry refuse with bedding materials enhanced plant growth and reduced root knot nematode disease incidence with higher fruits yield of tomato. This result is in agreement with that of Meyer et al. (38) who indicated significant increase in plant height, leaf area, shoot dry weight, stem girth and root dry weight of cacao plants with application of poultry manure. The application of poultry manure has been observed to have a suppressive effect on root-knot nematodes (39). Farahat et al. (40) also reported that efficacy of poultry manure against plant-parasitic nematodes may either be due to stimulation of specific micro-organisms that were capable of parasitizing eggs and juveniles or production of substances from decomposition of the manure which were toxic to the nematodes. From this study it was also observed that rice bran significantly enhanced plant growth and reduced root knot nematode incidence as well as increased fruits yield of tomato compared to untreated control. Chun-Yang et al. (41) reported that rice bran has frequently been utilized in the past for soil improvement. Chun-Yang et al. (41) also observed that rice bran ash reduces the plasticity of soils and increased organic matter content. In addition, rice bran was observed to have the ability to reduce plant-parasitic nematode infestation on crops. According to Prakash and Singh (42), nematode density and reproduction decreased progressively with increasing rice bran amendments. Thus it was revealed from the present investigation that soil amendment with poultry refuse was the most effective option for reducing root knot nematode disease and increasing plant growth as well as for higher yield of tomato. The other options were soil treatment with chemical pesticide Furadan 5G or rice bran for reducing root knot disease and increasing plant growth as well as for higher yield of tomato. Similar studies done by Hassan et al. (43) and Orisajo et al. (35) showed that amending the soil with organic waste materials such as poultry refuse, rice bran and saw dust suppressed the populations of Meloidogyne spp. both in the soil and roots of tomato with simultaneous increase in the growth and yield. Oka et al. (35) and Orisajo et al. (44) also reported that application of soil organic amendments is not only beneficial to nematode management but also to plant growth and productivity. Therefore, it may be concluded that soil amendment with poultry refuse is the best treatment followed by soil treatment with chemical pesticide Furadan 5G or rice bran for reducing root knot nematode disease and increasing plant growth as well as yield of tomato.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy:
Agricultural And Food Sciences
Environmental Science

text:
Drought-Induced Regulatory Cascades and Their Effects on the Nutritional Quality of Developing Potato Tubers

Competition for scarce water resources and the continued effects of global warming exacerbate current constraints on potato crop production. While plants’ response to drought in above-ground tissues has been well documented, the regulatory cascades and subsequent nutritive changes in developing tubers have been largely unexplored. Using the commercial Canadian cultivar “Vigor”, plants were subjected to a gradual drought treatment under high tunnels causing a 4 °C increase in the canopy temperature. Tubers were sampled for RNAseq and metabolite analysis. Approximately 2600 genes and 3898 transcripts were differentially expressed by at least 4-fold in drought-stressed potato tubers, with 75% and 69% being down-regulated, respectively. A further 229 small RNAs were implicated in gene regulation during drought. Expression of several small RNA clusters negatively correlated with expression of their six target patatin genes, suggesting involvement in the regulation of storage proteins during drought. The comparison of protein homologues between Solanum tuberosum L. and Arabidopsis thaliana L. indicated that down-regulated genes were associated with phenylpropanoid and carotenoid biosynthesis. As is indicative of reduced flow through the phenylpropanoid pathway, phenylalanine accumulated in drought-stressed tubers. This suggests that there may be nutritive implications to drought stress occurring during the potato tuber bulking phase in sensitive cultivars.

Introduction
Potatoes are the fourth most consumed food crop worldwide and are an efficient source of energy, vitamins and minerals in the human diet [1]. High consumption rates and moderate concentrations of dietary antioxidants have led potatoes to be the third-largest source of total phenolics in the American diet [2]. Diets rich in phenolics have been implicated in the prevention of an array of degenerative diseases and concentrations of these compounds vary greatly based on the cultivar, highlighting the potential for the targeted breeding of potato (S. tuberosum L.) to enhance global human health [3]. In potato tubers, the primary polyphenol is chlorogenic acid with the remaining components Saskatchewan. The cultivar "Vigor", a cross between "Agria" and "Wischip" from the Agriculture and Agri-Food Canada Lethbridge Research and Development Centre, was evaluated for its performance under soil moisture stress during the tuber bulking phase. Prominent characteristics of the cultivar are its yellow-fleshed tubers and pigmented (red-violet) flowers. Plants were grown under optimum soil moisture conditions at 70% field capacity (FC) and restricted soil moisture conditions at 35% FC under two high tunnels using drip irrigation. Treatments were imposed at the start of the tuber bulking phase for gradual exposure to drought stress, thereby mimicking natural field conditions (Figure 1). High tunnels were opened from all sides but covered with plastic film on top to mimic the open field condition while preventing rainfall (Supplementary Figure S1A). Plots were laid out in a randomized complete block design containing four replicates with guard rows on either side. Each plot consisted of 12 hills. The two end hills were considered as guard hills for yield estimation purposes. Seed pieces were spaced 1 m between-rows and 20 cm within-rows and were planted on 30 May 2017. The crop was raised using standard management practices (i.e., fertility, irrigation, pest control, etc.). Pre-plant basal fertilizer included urea (46-0-0), mono-ammonium phosphate (11-52-0), and potash (0-0-60). Two applications of ammonium sulphate (21.5-0-0-4) were given at 4 and 7 weeks after planting. Soil moisture was monitored using Watermark sensors (Supplementary Figure S1B). Plots were harvested on 2 October 2017 and graded according to commercial-grade standards.
Genes 2020, 11, x FOR PEER REVIEW 3 of 13 541 m), Saskatchewan. The cultivar "Vigor", a cross between '"Agria" and "Wischip" from the Agriculture and Agri-Food Canada Lethbridge Research and Development Centre, was evaluated for its performance under soil moisture stress during the tuber bulking phase. Prominent characteristics of the cultivar are its yellow-fleshed tubers and pigmented (red-violet) flowers. Plants were grown under optimum soil moisture conditions at 70% field capacity (FC) and restricted soil moisture conditions at 35% FC under two high tunnels using drip irrigation. Treatments were imposed at the start of the tuber bulking phase for gradual exposure to drought stress, thereby mimicking natural field conditions (Figure 1). High tunnels were opened from all sides but covered with plastic film on top to mimic the open field condition while preventing rainfall (Supplementary Figure S1A). Plots were laid out in a randomized complete block design containing four replicates with guard rows on either side. Each plot consisted of 12 hills. The two end hills were considered as guard hills for yield estimation purposes. Seed pieces were spaced 1 m between-rows and 20 cm within-rows and were planted on 30 May 2017. The crop was raised using standard management practices (i.e., fertility, irrigation, pest control, etc.). Pre-plant basal fertilizer included urea (46-0-0), mono-ammonium phosphate (11-52-0), and potash (0-0-60). Two applications of ammonium sulphate (21.5-0-0-4) were given at 4 and 7 weeks after planting. Soil moisture was monitored using Watermark sensors (Supplementary Figure S1B). Plots were harvested on 2 October 2017 and graded according to commercial-grade standards. Figure 1. Visualization of potato growth stages and in-season soil moisture trends when soil moisture was maintained at 35% and 70% field capacity (FC).

Physiological Measurements
Physiological measurements were taken at the end of the tuber bulking phase (90th day after planting) with readings recorded consistently between 11:30 a.m. and 12:30 p.m. Canopy temperature was assessed with an infra-red thermal imaging camera (FLIR T530, FLIR Systems. Wilsonville, OR, USA), and leaflet chlorophyll content (CCI) was recorded using a chlorophyll content meter (CCM-200-Apogee Instruments, Logan, UT, USA). Quantum yield of dark-adapted leaflets (Fv/Fm) was measured using the portable fluorometer FluorPen FP 100 (PSI, DRASO Czech Republic). Detachable clips were used to dark-adapt the leaflets for 20 min, and Fv/Fm was measured on the adaxial surfaces of the top 3rd and 4th leaflet of each sampled plant (three plants per replication).

Physiological Measurements
Physiological measurements were taken at the end of the tuber bulking phase (90th day after planting) with readings recorded consistently between 11:30 a.m. and 12:30 p.m. Canopy temperature was assessed with an infra-red thermal imaging camera (FLIR T530, FLIR Systems. Wilsonville, OR, USA), and leaflet chlorophyll content (CCI) was recorded using a chlorophyll content meter (CCM-200-Apogee Instruments, Logan, UT, USA). Quantum yield of dark-adapted leaflets (Fv/Fm) was measured using the portable fluorometer FluorPen FP 100 (PSI, DRASO Czech Republic). Detachable clips were used to dark-adapt the leaflets for 20 min, and Fv/Fm was measured on the adaxial surfaces of the top 3rd and 4th leaflet of each sampled plant (three plants per replication).

Amino Acid Profiling and Abscisic Acid Content
In both high tunnels from each replication, a tuber was collected from the middle of the plot on 13 September 2017 (106 DAP). The tubers were washed in running water, followed by distilled water, cut into cubes while evading the skin and immediately frozen in liquid nitrogen. Samples were stored at −80 • C until further use.
Amino acids were extracted from 10 mg of ground freeze-dried tissue following Inaba et al. (1994) [19] with some modifications. Briefly, 1 mL of 80% (v/v) ethanol solution (40 • C) was added to each sample, shaken for 30 min at 40 • C and the supernatant was recovered by centrifugation (4000 rpm for 10 min) at 4 • C. The pellets were re-extracted under the same conditions with an additional 500 µL of 80% (v/v) ethanol (40 • C). The supernatants were combined and stored at −20 • C until further use. Amino acids were derivatized following Waters AccQTag Reagent Kit (Waters, Milford, MA, USA; [20]). Briefly, a 10 µL aliquot of sample was mixed with 70 µL borate buffer and 20 µL AccQFluor reagent which was reconstituted in acetonitrile. AccQFluor reagent was reconstituted as follows: 1 mL of AccQFluor reagent diluent was transferred to a vial containing AccQFluor reagent powder and vortexed for 10 s before heating at 55 • C for a maximum of 10 min or until dissolved. The derivatized mixture was transferred to an autosampler vial and incubated at 55 • C for 10 min. High-performance liquid chromatography (HPLC) was conducted, as described in Waters AccQTag's chemistry package instruction manual, with samples separated on a Waters amino acid column −3.9 × 150 mm and quantified at an excitation wavelength of 285 nm and an emission wavelength of 320 nm using a 2475 scanning fluorescence detector (Waters, Milford, MA, USA). The column was set at 37 • C with a 5 µL injection volume. Waters AccQTag buffer (100 mL AccQTag Buffer concentrate +1000 mL deionized water), acetonitrile, and deionized water were used as mobile phases A, B, and C, respectively.
Abscisic acid content was determined following Yan et al., (2016) [21]. Samples were centrifuged to remove debris, and the pellet was washed twice. The supernatant was evaporated in a SpeedVac, and reconstituted in 1 mL of 1% (v/v) acetic acid. Abscisic acid (ABA) was purified by solid-phase extraction using Oasis HLB, MCX, and WAX cartridge columns (Waters, Milford, MA, USA). The solvent was removed under vacuum and subjected to LC-ESI-MS/MS analysis (Agilent 6410 TripleQuad LC/MS system). An LC (Agilent 1200 series) equipped with a 50 × 2.1 mm, 1.8-µm Zorbax SB-Phenyl column (Agilent Technologies, Santa Clara, CA, USA) was used with a binary solvent system comprised of 0.01% (v/v) acetic acid in water (solvent A) and 0.05% (v/v) acetic acid in acetonitrile (solvent B). Separations were performed using a gradient of increasing acetonitrile content at a flow rate of 0.2 mL min −1 . The gradient was increased linearly from 3% B to 50% B over 15 min. The retention time of ABA was 14 min.

Transcriptome and Small RNA Sequencing
Total RNA was extracted from 100 mg of tuber tissue partitioned from the sample taken for metabolite analysis using the RNeasy plant mini kit (Qiagen, Hilden, Germany). RNA quality and concentration were verified using an Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). TruSeq RNA and small RNA sequencing libraries were constructed following the standard preparation guide (Illumina, San Diego, CA, USA). All eight RNA samples (four replicates of each treatment) were multiplexed in a lane of a flow cell and paired-end sequencing (125 cycles) was performed using an Illumina HiSeq 2500. Similarly, for small RNA sequencing, all 8 samples were multiplexed in a lane of a flow cell and single-end sequencing was carried out on Illumina HiSeq 2500.

RNA and Small RNA Read Mapping and Analysis
Before read mapping and expression quantification, all RNA reads were filtered using Trimmomatic (version 0.36; [22]) by (i) removing adapter sequences, (ii) trimming leading and trailing low-quality sequences, (iii) removing sequences when the average quality per base dropped below 15 within a 4-base wide sliding window and (iv) keeping only those pairs where both reads were longer than 75 bp. Clean reads were aligned to the potato reference genome (SolTub_3.0, EnsemblPlants) with STAR (v2.5.2b) and isoform expression was quantified with the RSEM (v1.3.3) algorithm [23]. The expected read counts generated by the RSEM algorithm were rounded off and fed into DESeq2.

Differential RNA Expression Analysis
Raw read counts obtained from RNAseq were normalized and assessed for differential expression using the Statistical Software "R" version 3.6.0 and the package DESeq2 [28,29]. Log2 fold change threshold of 2 and a 5% false discovery rate (FDR) were used as cut-off values for continuing to the annotation step. R scripts are available on Bioconductor (https://bioconductor.org/) from the package developers and were adapted for the data presented in this paper. The same technique was repeated for the discovery of differentially expressed small RNA, with target gene identification done using psRNATarget [27]. Gene annotations for S. tuberosum and A. thaliana were obtained from the Ensembl Plants database (http://plants.ensembl.org). Arabidopsis homologs with >50% identity to the original potato gene were input into the online DAVID Bioinformatics Resources version 6.8 (https://david.ncifcrf.gov/) for functional annotation clustering and KEGG pathway mapping analyses.

Physiological Response
A high degree of variability existed within the four control and four treatment plots for the agronomic and physiological traits measured during this study (Table 1). Both crop yield and tuber number per plot were not found to be significantly different from one another, although differences in plot averages were observed. Calculated values from spectral measurements, such as CCI and Fv/Fm, showed no significant differences. However, canopy temperatures measured in plots maintained at 35% FC were 3.9 • C higher than control plots with soil moisture maintained at 70% FC (Table 1). ABA concentrations in the well-watered control and water-deficit conditions were 43.9 and 55.4 ng gDW −1 , respectively. Variation was high among the control plants, resulting in no significant differences between treatments.

Tuber Amino Acid Fluctuations in Response to Soil Moisture Deficit
Of the eight essential amino acids, lysine, phenylalanine, isoleucine, and leucine were found to be more abundant in drought-stressed tubers. The largest differences were observed in the concentrations of leucine, phenylalanine, and isoleucine which increased by 3×, 2×, and 1.9×, respectively (Figure 2A). Quantities of branched-chain amino acids, a group that includes leucine, isoleucine, and valine, were therefore significantly higher under drought treatment. Histidine and valine were the most abundant essential amino acids in developing potato tubers (Figure 2A). The majority of non-essential amino acids had similar concentrations in developing tubers regardless of the treatment. Only glutamic acid showed a marked increase of 5.75 µmol g −1 under reduced soil moisture conditions. Concentrations of cysteine, proline, and serine were highest among all amino acids measured ( Figure 2B).
Genes 2020, 11, x FOR PEER REVIEW 6 of 13 the treatment. Only glutamic acid showed a marked increase of 5.75 µmol g −1 under reduced soil moisture conditions. Concentrations of cysteine, proline, and serine were highest among all amino acids measured ( Figure 2B).

Figure 2.
Concentrations of essential (A) and non-essential (B) amino acids in potato tubers (n = 4) sampled during the tuber bulking phase and the associated SEM (p < 0.05) when subjected to 70% and 35% field capacity.

Differential Gene Expression and Regulatory Cascades in Developing Tubers Under Drought Stress
Tubers subjected to soil moisture deficit showed both differential gene and transcript expression, with 75.2% and 68.8% being down-regulated, respectively. One-fifth of genes with differential sampled during the tuber bulking phase and the associated SEM (p < 0.05) when subjected to 70% and 35% field capacity. * significance (p < 0.05) between treatments.

Differential Gene Expression and Regulatory Cascades in Developing Tubers Under Drought Stress
Tubers subjected to soil moisture deficit showed both differential gene and transcript expression, with 75.2% and 68.8% being down-regulated, respectively. One-fifth of genes with differential expression were of unknown function. A full summary of the observed changes is listed in Figure 3A with a list of all differentially expressed genes provided in Supplementary Table S1. Down-regulated genes include those with functions in ABA, auxin and ethylene signaling as well as in auxin, carotenoid, and phenylpropanoid biosynthesis. Up-regulated genes have roles in amino acid biosynthesis, function as molecular chaperones and are involved in ubiquitin-driven proteolysis. Gene names, functional annotation, and the corresponding Arabidopsis homologs used for pathway mapping can be found in Table 2. Focusing on the regulation of the phenylpropanoid pathway, fifteen annotated MYB transcription factors were down-regulated by more than 4-fold under low-soil moisture conditions. Up-regulated transcription factors include two MYB transcription factors and one LIM transcription factor. No other MYB, KNOX or LIM transcription factors were above the cut-off values of 5% FDR and a log-fold change greater than two. Genes with key functions in the phenylpropanoid and carotenoid pathways that were down-regulated in potato tubers at 35% FC are highlighted in Figure 4. Raw read counts for gene expression analysis can be found in Supplementary Table S2. with a list of all differentially expressed genes provided in Supplementary Table S1. Down-regulated genes include those with functions in ABA, auxin and ethylene signaling as well as in auxin, carotenoid, and phenylpropanoid biosynthesis. Up-regulated genes have roles in amino acid biosynthesis, function as molecular chaperones and are involved in ubiquitin-driven proteolysis. Gene names, functional annotation, and the corresponding Arabidopsis homologs used for pathway mapping can be found in Table 2. Focusing on the regulation of the phenylpropanoid pathway, fifteen annotated MYB transcription factors were down-regulated by more than 4-fold under low-soil moisture conditions. Up-regulated transcription factors include two MYB transcription factors and one LIM transcription factor. No other MYB, KNOX or LIM transcription factors were above the cutoff values of 5% FDR and a log-fold change greater than two. Genes with key functions in the phenylpropanoid and carotenoid pathways that were down-regulated in potato tubers at 35% FC are highlighted in Figure 4. Raw read counts for gene expression analysis can be found in Supplementary  Table S2.
The isolated small RNA were grouped into 87,213 clusters with an additional 10,209 unassigned sequences. Of these, 103 clusters and 126 unassigned sequences were differentially expressed. Additional summary statistics are listed in Figure 3B. Differentially expressed small RNA clusters with identified gene targets are listed in Supplementary Table S3. None of the small RNA clusters with target genes listed in Table 2 showed differential expression between the two treatments. Expression of target MYB transcription factor genes was also not correlated to small RNA cluster expression (Supplementary Table S4). Interestingly, the expression of small RNA clusters primarily targeting patatin genes was significantly up-regulated and negatively correlated to target gene expression (r = -0.61). These clusters and their targets can be found in Table 3.  thaliana were considered if identity was greater than 50%. Functional annotation clustering to KEGG pathways was based on Arabidopsis gene IDs using the DAVID Bioinformatics online resource 6.8 (https://david.ncifcrf.gov/). Table 2. List of differentially expressed genes in drought-stressed potato tubers, the corresponding homologs in A. thaliana, and the pathway in which they participate. Pathway mapping was done based on the Arabidopsis gene names and similarities between the original S. tuberosum gene and its homolog are expressed as the percentage of identical base pairs in the gene sequences (% ID).   significantly suppressed under drought (Supplementary Table S1). Unlike previous findings in potato leaves [10], expressions of small RNA and their target MYB transcription factors were not correlated in the drought-stressed tubers. There was therefore no evidence to suggest that small RNA played a role in regulating the phenylpropanoid or carotenoid pathways under drought conditions in potato tubers (Supplementary Table S4).

Conclusions
Potato is among one of the most important food crops, yet maintaining plant productivity in this drought-sensitive crop has become a challenge. From a nutrition perspective, decreasing soil water availability during tuber filling, as a function of a warming climate or as a production practice to induce senescence for an earlier harvest, may lead to a reduction in tuber quality. While mild drought increases the proportion of essential amino acids, potential losses in protein and phenolic content would outweigh the benefit. While MYB transcription factors may be targeted to reduce effects on the phenylpropanoid pathway, identification of small RNA as the regulator of patatin gene expression suggests it may be difficult to maintain patatin expression in drought-susceptible cultivars using current breeding techniques.
Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1; Figure S1: Experimental conditions;   The isolated small RNA were grouped into 87,213 clusters with an additional 10,209 unassigned sequences. Of these, 103 clusters and 126 unassigned sequences were differentially expressed. Additional summary statistics are listed in Figure 3B. Differentially expressed small RNA clusters with identified gene targets are listed in Supplementary Table S3. None of the small RNA clusters with target genes listed in Table 2 showed differential expression between the two treatments. Expression of target MYB transcription factor genes was also not correlated to small RNA cluster expression (Supplementary Table S4). Interestingly, the expression of small RNA clusters primarily targeting patatin genes was significantly up-regulated and negatively correlated to target gene expression (r = −0.61). These clusters and their targets can be found in Table 3.

Discussion
Optimal potato tuber growth occurs around 20 • C and plants are susceptible to losses in productivity under hot, arid conditions. Such conditions are expected to increase in the coming decade, therefore functional indicators of plant stress and the cascading effects on the developing tubers were evaluated. In this study, the Canadian potato cultivar "Vigor" was gradually exposed to increasing water deficit to a level of 35% FC beginning at the start of the tuber bulking phase ( Figure 1). As seen previously in the literature, fluorescent measurements were not distinguishable between treatments [15] and thus were not dependable indicators of drought stress in potato plants (Table 1). At the plot level, there were no significant differences in yield or tuber number between the treatments (Table 1), however, discrepancies could become more prominent in commercial field production. Canopy temperature was considerably elevated in the drought treatments and has evidence supporting its use for drought stress assessments [30]. Average concentrations of ABA trended upwards in potato tubers exposed to water deficit compared to well-watered controls, suggesting drought responses had been initiated. Gene expression data further corroborated that drought signaling pathways had been activated as there was marked down-regulation of an ABA receptor PYR1, down-regulation of a series of small auxin up-regulated RNA (SAUR) genes involved in cell expansion and organ elongation in response to the environment [31] and the up-regulation of heat shock factor proteins [32] (Table 2). Notably, outside of common changes to the regulation of heat shock proteins, several genes previously identified as differentially expressed in severely drought-stressed potato leaves [32] were inversely regulated in the mildly stressed potato tubers collected in this study. These include the WRKY transcription factor (PGSC0003DMG400001434) and the developmental gene UPA16 (PGSC0003DMG400031742) [32] which had 2-fold and 78-fold increases in expression compared to well-watered control tubers. Lists of genes implicated in drought stress response in potato leaf tissue [33] and potato stolons [34] have been compiled and here we provide those in developing tubers (Table 2; Supplementary Table S1).
Metabolic effects of drought on parameters such as free amino acids, soluble protein, and phenolics were assessed. The gradual drought stress to which the potato tubers were exposed resulted in no significant differences in total free amino acid concentrations, although treatment averages appeared to be divergent with 154.1 and 207.2 µmol g −1 in the control and drought treatments, respectively. These differences could be associated with the up-regulation of genes involved in proteolysis (Table 2). Elevated concentrations of proline have been shown to indicate stress in potato leaves [35], however, similar concentrations were observed in tubers irrespective of treatment. The largest changes occurred in the amino acid profile, where concentrations of branched-chain amino acids leucine and isoleucine increased ( Figure 2). This indicated a greater proportion of dietary essential amino acids. Increases in branched-chain amino acids are likely attributable to the up-regulation of acetolactate synthase ( Table 2), which is the first enzyme in the branched-chain amino acid synthesis pathway [36].
A major fraction (up to 40%) of the soluble protein in potato tubers consists of glycoproteins, known as patatins, that act both as storage proteins and show activity as non-specific lipid acyl hydrolases (LAH) with potential roles in plant defense against biotic stressors [37,38]. In the case of abiotic stress, it was observed that five patatin genes were down-regulated by at least 16-fold with the regulation of gene expression likely occurring via an increased presence of small RNA (Table 3). A possible consequence is reduced protein content in the resulting potato tubers.
As one of the major sources of plant phenolics in the human diet, potatoes have been targeted in breeding for greater total phenolics and antioxidant capacity [3,39]. Phenolic content is known to show a high degree of environmental plasticity with cooler temperatures during the growing period and storage, attributed to higher average accumulation [4,5]. Under drought conditions, expressions of key enzymes required for the biosynthesis of anthocyanins (DFR), flavonoids (FLS) and chlorogenic acid (HCT) were drastically reduced ( Figure 4A). Initial flow into the phenylpropanoid pathway through PAL was also reduced, leading to the accumulation of phenylalanine observed in Figure 2. Similar results have been previously observed in the literature [5]. Key enzymes of the carotenoid pathway were also downregulated ( Figure 4B). Environmental conditions leading to the repression of phenolic biosynthesis could minimize gains achieved in breeding programs. Regulation of the phenylpropanoid pathway can occur via MYB transcription factors [8], fifteen of which were significantly suppressed under drought (Supplementary Table S1). Unlike previous findings in potato leaves [10], expressions of small RNA and their target MYB transcription factors were not correlated in the drought-stressed tubers. There was therefore no evidence to suggest that small RNA played a role in regulating the phenylpropanoid or carotenoid pathways under drought conditions in potato tubers (Supplementary  Table S4).

Conclusions
Potato is among one of the most important food crops, yet maintaining plant productivity in this drought-sensitive crop has become a challenge. From a nutrition perspective, decreasing soil water availability during tuber filling, as a function of a warming climate or as a production practice to induce senescence for an earlier harvest, may lead to a reduction in tuber quality. While mild drought increases the proportion of essential amino acids, potential losses in protein and phenolic content would outweigh the benefit. While MYB transcription factors may be targeted to reduce effects on the phenylpropanoid pathway, identification of small RNA as the regulator of patatin gene expression suggests it may be difficult to maintain patatin expression in drought-susceptible cultivars using current breeding techniques.


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Cultivation technology and antibacterial activity of Agaricus bisporus (U-03)

Agaricus bisporus, being one of the most commonly white button mushrooms contributes about 4045% to the world mushroom production. Presence of viable cells were measured in terms of viable count or CFU, which exhibit progressive declination in count of thermophilic fungi from initial stage of composting. Similarly, declination of pH ranges from 8.3-7.1 alkaline pH in initial stage was due to microbial decomposition of organic acids and subsequent release of ammonia through mineralization of organic nitrogen sources, declination of weight from 1.465 Kg. to 0.491 Kg and temperature from 44-34◦C was due to extensive utilization of compost as nutrient by inhabiting thermophilic fungi. Result obtained exhibit highest cellulose content in raw material of which horse manure exhibit higher cellulose content then wheat straw, horse manure 2.052mg and wheat straw 1.044mg. Highest cellulolytic activity was observed in horse manure followed by wheat straw in basal material. Horse manure 2.167micromoleglucose released/ml/hr and wheat straw 1.014micromoleglucose released/ml/hr. Highest antimicrobial activity was observed in methanol extract (14mm) against B. cereusas compared to ethanol extract (7mm). Same results obtained for S.aureus(20mm) and B. cereus (14mm) in methanol extract. Least microbial growth inhibition was observed in S. aureus (25mm) followed by B. cereus (7mm) with respect to control in ethanol extract. No antimicrobial activity observed in P. aeruginosa in ethanol but least inhibition was observed in methanol extract. © 2020 Published by Innovative Publication. This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/)

Introduction
Agaricus bisporus, being one of the most commonly white button mushrooms contributes about 40-45% to the world mushroom production (Flegg, 1992). A. bisporus is a low temperature species requiring 23 ± 20 C for its vegetative growth and 16 ± 20 0 C for its fruiting (Singh, 2011) and widely consumed mushrooms in the world is cultivated in more than seventy countries. Morphologically, A. bisporus bears 3-16 cm cap, convex to broadly convex, dry, smooth or with pressed-down fibers or small scales, white in some varieties, brown in others, with gills which are free from the stem, close, pinkish to pinkish brown at first, becoming dark brown to blackish. The stem measures around 2-8 cm long, 1-3 cm. thick, study, more or less equal, smooth or with small scales below the ring, white, often bruising brownish, with a ring that sometimes disappears in maturity. A. bisporus flesh is white and firm, usually bruising and staining brownish, and give pleasant odor and taste. Microscopically, the brown spore print exhibits as spores 5.5-8.5 x 4-6.5 µ, elliptical, smooth with 2-spored basidia (kuo, 2004). A.bisporus mushrooms are well-known for their nutritional and medicinal values. They are also valued for waste management as most of them grow on lignocellulosic materials of agricultural origin, forest litter and garden litter. A. bisporus is a source of protein (about 40% on dry basis), ergo sterol, a precursor of vitamin D, several minerals, carbohydrate and fat. The major minerals detected in it are sodium, potassium and phosphorus. Agaricus bisporus has substantial amounts of Vitamin D which is good for our health. Eating the mushroom raw is discouraged because of the presence of carcinogenic hydrazine derivatives, albeit in small quantities. A. bisporus mushroom contains Potassium and Sodium. The diet dependent statistically significant decrease of incidence of the disease has been reported in women diagnosed with breast cancer. It exhibit anti-microbial properties and antioxidant properties as evidenced by inhibitory properties of its extract against some gram positive as well as gram negative bacteria. Different methodologies such as microdilution method, the disk diffusion method, the agar streak dilution method based on radial diffusion method and a method with the incorporation of the extract in culture medium and further determination of colonies have been used to asses antimicrobial activity of A. bisporus extracts and compounds. [1][2][3] The above cited description points to the nutritional and medicinal values and subsequent studies on Agaricus bisporus with reference to its cultivation and antimicrobial properties however, there is hardly such report available in the vicinity of industrial hub of Himachal Pradesh. Thus, present piece of work highlights the mushroom cultivation under prevalent environmental conditions using regional resources and local bacterial isolates. (Singh, 2006) Under aseptic conditions using laminar flow, young basidiocarp was cleaned with sterilized distilled water and dipped into 0.1% mercuric chloride or 2.5% sodium hypochlorite solution for 1 min or washed with 85% ethanol and air dried then button mushroom split open longitudinally from centre and vegetative mycelial bits are cut from the collar region (junction of pileus and stipe) after that bits are inoculated centrally in starch/Malt extract agar (MEA) plates and inoculated plates are incubated at 25±2 0 C in a BOD incubator. Within 4-5 days the new mycelium growing over the media was observed. The pure cultures were made by carefully transferring the young mycelium from growing edge of the colony from petriplates to test tubes and again incubated at 25±2 0 C for 10-14 days. Three replica of each media were maintained. 4-6 (Singh, 2006) The full growth culture tubes were stored at cooler temperatures, i.e., at 4 0 C to 7 0 C in a refrigerator or cold room till further use. (Vijay, 1996) About 300 g prepared substrate (boiled wheat grains mixed with gypsum and chalk @ 2.0% & 0.5%) was filled in Erelenmeyer flasks upto 2/3 volume and plugged with nonabsorbent cotton. The plugs are covered with aluminum foil. These filled bottles were autoclaved at 15 psi pressure.

Spawn preparation
After autoclaving bottles were left in the room for 24-48 hrs for cooling. A piece of mycelium (pure culture) grown in Petri plates was aseptically transferred to these bottles and inoculated bottles were incubated at 25±1 0 C. These bottles are gently shacked on 5 th and 10 th day for distributing the inoculum evenly in the bottles. After 10 th day grains were fully colonized with button mushroom mycelium (U-03). The fully colonized grains were then used as spawn or inoculants in compost. Wheat straw was dip in tub containing water having trace amount of formalin and bavistin in it, for 24-48 hrs to achieve 70-73% moisture. After soaking all ingredients were mixed in wheat straw and then mixed composite materials were filled in sterile beakers @ 1 Kg. Whole sets were incubated at 47±1 0 C and after filling periodic turning of 13 and 12 were given to all the sets. So that, malodorous gases get eliminate from the compost and compost become aerobic which made possible to grow aerobic microorganism's mainly thermophilic microorganisms in compost. This made compost selective for the growth of A, bisporus. (Vijay, 1996) 2.5.1. Spawning After completion of composting, compost of both sets were spawned with desired inoculants (A. bisporus, U-03). The spawned compost of both sets was incubated at 25±1 0 C for 12-15 days. After completion of spawn run, a layer of sterile casing made up of 1:3 ratio of farmyard manure and coir pith were placed on the top surface of the compost which facilitate the fruiting bodies formation. After casing again both sets of compost were incubated at 25±1 0 C till the mycelium of compost emerges to the casing layer. After that small pin head structure of nascent mushroom fruiting bodies get appeared on the casing layer. Fruiting bodies formation takes place after 7-8 days of casing.

Harvesting
When fruiting bodies appeared on the compost bag as pin heads the temperature of the compost bag were made lower down to 18±1 0 C, humidity 80-90% and ample of fresh air were introduced to the sets. At this temperature mushroom develop which later on get harvested by gently twisting. After harvesting yield were recorded on digital balance.

pH
Ten grams of composite sample of each set was suspended in 100 ml. of distilled water and shaken for some times. The pH of suspension was determined by using digital pH meter.

Temperature
The temperature of both sets was daily recorded with the help of mercury thermometer.

Weight loss
Weight loss as weight of beakers was recorded from filling to end of composting and is estimated on digital pan balance and calculated by using formula as given below. After that test tubes were cooled at room temperature and then centrifuged at 10,000 rpm for 15-20 min. After centrifugation supernatant were discarded and residues were washed with distilled water. Then after 10 ml. of 67% concentrated sulphuric acid were added in each test tube and allowed the test tubes to stand at room temperature for 1h. Now one ml. of above solution were taken in other test tube and diluted by adding 99 ml. of distilled water in it. Again one ml. of diluted sample were taken in other test tubes and 9 ml. of anthrone reagent were added in it. After adding anthrone reagent test tubes were boiled in water bath for 10 min. cooled and then measure the colour at 630 nm. Blank containing 1 ml. distilled water and 9 ml. anthrone reagents were used as control. Estimation of cellulose was done by making standard curve corresponding 40-200µg. of cellulose and then calculate the cellulose % by using formulae given below. Screening of anti bacterial activity property of A. bisporus.

Total cellulose =
Isolation and identification of gram positive and gram negative bacteria from sewage sample 2.12. Collection of samples: (Willey, Sherwood and Woolverton 2011 For the isolation of different gram positive and gram negative bacteria sewage samples were collected from different sewage treatment plants in sterile beaker and collected samples were transferred to sterile plastic bags in aseptic conditions.

Procedure
Serial dilution agar plating method is one of the most commonly used procedure for the isolation of microorganisms from sewage samples. In this 1ml of sewage sample was added to 9ml of distilled water to make the final volume 10ml. Serial dilutions were done ranging from10 −2 , 10 −3 ,. . . . . . .10 −7 by pipetting 1ml into additional dilution blanks. Finally, 1ml aliquot of various dilutions was added to sterile petriplates having 15ml of sterile, cool, molten nutrient agar media. The dilutions 10 −4 to 10 −7 were selected for enumeration of bacteria. Upon solidification, the plates were incubated in inverted position for 3-4 days at 37±1°C after incubation, different types of colonies were appeared on plates. Three replication of each dilution were maintained.

Identification of microorganisms: (Holt et al, 1994)
After inoculation the isolated colonies appeared on medium were identified on the basis of Bergy's mannual of determinative bacteriology. The isolated microorganisms were identified on the basis of colony morphology, texture, Gram staining, Endospore staining and biochemical tests.

Colony morphology
Different types of isolated microorganisms exhibited different types of colonies on surface of agar which were later identified externally by seeing characters like shape (circular, dot like, irregular etc.), colony elevation (thin film, raised, convex etc.), colony margins (entire, crenate, dentate, lobate etc.), optical density (transparent, tanslucent, opaque etc.).

Texture
Slimy and glistering colonies were seen on the medium.

Oxidase test
Oxidation and fermentation glucose media(OF glucose media). was autoclaved in tubes and then cooled for some time. One tube was inoculated with isolated bacterial culture and other tube was not inoculated which was used as a control. Then liquid paraffin was poured over the medium to form a layer about 1cm in depth in the tubes. Then tubes were incubated at 35±1 0 C for 24-48 hrs. Composition of medium is given in Appendix.

Mannitol fermentation test
Mannitol salt agar media plates were prepared and streaked with isolated bacterial culture. These plates were incubated at 37±1 • C for 24-48 hrs. Composition of given medium is given in Appendix.

Purification and preservation of isolated microorganisms: (Willey, Sherwood and Woolverton 2011
Isolated and identified colonies were maintained on slants and kept at 4±1 0 C till further use. Slants were revived periodically to maintain the colonies in active conditions.

Screening of anti-bacterial activity of A bisporus 2.20.1. Collection of A. bisporus(U-03)(Vijay, 2006).
Fresh and healthy fruiting bodies of A. bisporus (U-03) were collected from the beakers and after that fruitbodies were kept at 47±1 0 C in hot air oven to get fully dried till constant dry weight.

Preparation for extract(Sagar et al., 2012).
Dried fruiting bodies of A. bisporus(U-03) were weighed to know the moisture content and then grinded in fine powder in mortar pestle. Powder of one gram dried fruit bodies were suspended in 9 ml. ethanol, methanol and distilled water. Extraction was accomplished by stirring solution at 150 rpm for 24h at 20 0 C. The broth was then centrifuged at 10,000rpm for 10 minutes and the supernatant was filtered by using whatman No. 1 filter paper and extracts were collected in pre sterilized micro-centrifuge vials. Extracts were then stored at 4 0 C for study of antibacterial activity.

Screening of anti-bacterial activity of A. bisporus(U-03)
. (Bauer,. et al., 1966) In vitro anti-microbial susceptibility tests are performed by using identified pathogenic microorganisms following agar well diffusion method. NAM was used throughout the investigation for the growth of microorganisms. Medium was autocalved at 121 0 C for 30 minutes. The plates were left overnight at 37±1 0 C for pre incubation to check for any contamination to appear. Then after freshly grown bacteria were seeded on the medium to prepare bacterial lawns. Inoculated plates were incubated at 37 0 C for 24 hrs. to develop lawn on the plate and then after two agar wells of 8mm diameter were made opposite to each other. One well serves as control and other serve as test. 10 µl. of extract were pipetted in one well and its and its respective solvent without extract were pipetted in other well. After pipetting plates were kept at 20±1 0 C for 1 hrs. so that extract get absorb in the medium. After that plates were incubated at 37±1 0 C for 24 hrs. to see the effect of extract on bacterial lawn and result thus obtained get compared to control treatment.

Compost preparation and its physiochemical and biological parameter
Compost is the substrate used for cultivation of A. bisporus.
A. bisporus utilize compost as nutrient source carbohydrate and protein part of compost serve as carbon and nitrogen source for A. bisporus. A. bisporus compost was prepared by using ingredients and methods as mention in para 1d.of section A of materials and methods. Moisture of compost was adjusted to 70-73% and composting was done under in vitro condition or lab condition. About one kg. of compost was filled in pre weighed beakers and set of filled beakers were incubated at 47+1 0 C for 12-13 days. Periodical turnings (12-13 turnings) of compost were given from zero day(filling day) to maturation stage (at the time of spawning) and compost get matured or get ready for spawning after 21 days. Turning is given after two days interval from filling so that ammonia and other malodorous gases generated due to fermentation must get escape and make compost aerobic so that proliferation and colonization of thermophilic microorganism get well established in compost. Dominance of these fungi in compost at early stage of composting make compost nutritive and selective for the growth of A. bisporus. Several workers also reported the dominance of thermophilic microorganisms in compost in early stages which favors the growth of A. bisporus in compost. (Waksman et al.,1939, Vijay, 1996, Pathak, 2014. These thermophilic microorganisms utilize cellulose and proteins as carbon and nitrogen. Thermophilic microorganisms get carbon and nitrogen sources from agricultural residue and manure supplemented at the time of composting as raw material. Lille amount of Urea and gypsum is also added in compost. Addition of this component in compost maintains the C/N ratio and pH of compost. Due to the presence of high amount of carbohydrate and protein compost become favorable of inhabiting microorganism. In this case thermophilic microorganisms well colonized in early stage of composting and later starts decline. Declination in population is due to the saturation or less availability of limited nutrition in compost. Result as dominance or presence of viable cells were measured in terms of viable count or CFU. Count of thermophilic fungi gradually decline from initial stage to final stage of composting. Similarly, declination in pH, weight and temperature of compost were also observed from initial to final stage of composting. Declination of pH ranges from 8.3-7.1 alkaline pH is due to the microbial decomposition of organic acids and subsequent release of ammonia through mineralization of organic nitrogen sources. Rapid declination of Ph from initial to final stage of composting is due to the utilization of animal manure and nitrogen fertilizer in the compost and moreover scarification of compost which result in neutral pH and sweet smell of the compost. Compost at this pH favors the growth of A. bisporus hence spawning or adding inoculants at this pH is recommended. Hence at this stage U-03 was spawned in compost. Declination of weight from 1.465 Kg. to 0.491 Kg. and temperature from 44 -34 0 C is due to extensive utilization of compost as nutrient by inhabiting thermophilic fungi. Result observed in temperature study was quite surprising in initial stage of composting temperature of compost was low later or at room temperature later on it started rising and maximum in fourth turning (44 0 C) then after it started stabilizing and it remains stable till seventh turning then after it started declining and stabilized at maturation or twelfth turning(34 0 C). The up and down fall in temperature is thought to be of gases accumulated in compost during composting and respiration of thermophilic fungi population in compost which get released during turning. Several workers also reported the same (Vijay, 1996, Sharma, 1991, Satyanarayanan, 1987. Result as CFU count, pH, Weight loss and temperature is depicted in given (Table 1). Hence to know the succession of cellulose or total amount of cellulose present in compost from first turning to final stage of composting and during spawn run a cellulose estimation was performed in basal raw material (Wheat straw & horse manure), in different turnings of compost and during spawn run by using standard protocol given in para 3a(i) in chapter-2. Result obtained exhibit highest cellulose content in raw material of which horse exhibit higher cellulose content then wheat straw. Cellulose concentration in compost measured at the time of first turning was high as compared to first, sixth and tenth turnings of compost but low as compared to raw material. Lowest concentration of cellulose in each successive turning as compared to basal material is due to the utilization of cellulose by pre dominant microorganisms present in compost during composting and A. bisporus in mature compost. Degradation of cellulose during composting make readily available sugar in substrate which later used by A. bisporus for their growth. Result as total cellulose concentration of raw material and in compost is calculated on the basis of standard graph. Result given in Tables 2, 3 and 4.

Standard cellulose
A straight line plotted on standard graph at different concentration of cellulose. Intersection appeared on each point of cellulose concentration. Optical density of std.3 was later used for calculation of total cellulose concentration ( Table 2). Total cellulose concentration on basal material, different succession of composting and during spawn run.

Cellulase assay
Cellulose is commonly degraded by an enzyme called cellulase. Cellulase is secreted in compost by inhabiting thermophilic microflora. The end product of cellulose is glucose. Thus release of glucose in basal material and compost was estimated by Filter paperase method (FPase) or exo glucanase by using standard protocol (Miller, 1959). Detail methodology of protocol is given in ---of chapter 3. Similar trend as total cellulose concentration was observed in cellulase assay. Highest cellulolytic activity was observed in horse manure followed by wheat straw in basal material. Downward fall of cellulolytic activity was exhibited in different turnings of composting and least was exhibited during spawn run. Hence from the observation it was concluded that horse manure is the chief source of glucose which was readily utilized during composting by different thermophilic fungi this results in progressive down fall of glucose in compost. However small elevation of glucose concentration was observed in second turning it was due to the highest degradative activity of densely populated thermophilic fungi. After second turning declination of FPase activity was exhibited. Declination of glucose concentration adversely affect the population of growing thermophilic fungi which results in low count of CFU from I st turning to spawning, But this will not effect on growing of button mushroom as button mush room take nutrition from thermophilic microflora and degraded straw based material. Result as micromole glucose released/ml/ hr in basal material as well as in compost at different turnings are given (Tables 5, 6, 7 and 8).

Anti microbial activity of A. bisporus (U-03)
Screening of anti microbial activity of U-03 was observed on two bacterial isolates which were isolated from sewage samples and identified on the basis of morphology, Gram staining and biochemical properties. Detail methodology of antimicrobial assay is gtiven in para--chapter 2. Result as identification of bacterial isolates and inhibition zone formation by U-03 is given (Tables 7 and 8).
From the Table 8 it is clear that highest inhibition was observed in methanol extract as compared to ethanol extract. Highest inhibition was observed in plate inoculated with S. aureus, p.aeruginosa and B. cereus in methanol extract. Least inhibition was observed in S. aureus followed by B.cereus. with respect to control in ethanol extract.

Conclusion
From the present investigation it was concluded that agaricus bisporus (U-03) was cultivated on aerobic fermented substrate known as compost. Fermentation in compost was brought by inhibiting thermophilic microflora in it. During fermentation various physiochemical and biological changes were observed it was due to the utilization of substarte as nutrient sources by thermophilic fungi. Colonization of this fungus made compost favourable for the growth of U-03 in compost. U-03 extensively proliferates in compost by utilizing thermophilic microflora along with compost as nutrition sources. Time taken for compost production was 21 days and colonization of mushroom mycelium on compost 7-10 days. Later on investigation was done for screening of antibacterial activity of U-03 against three bacterial isolates viz B. cereus, S. aerus and P. aeruginosa and it was found that the extract of this mushroom was more or less inhibitory to all three bacterial isolates. Hence there is need to exploitation of U-03 along with thermophilic fungi on large scale for

Source of Funding
None.

Conflict of Interest
None.


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Drying kinetics and thermodynamic properties of bitter melon (Momordica charantia L.) leaves

ABSTRACT Bitter melon (Momordica charantia L.) is a versatile plant that can be consumed as a food and has therapeutic applications. Studying its drying process is important to maintain their leaf quality during storage. The objective of this study was to evaluate the drying kinetics of bitter melon leaves and determine their thermodynamic properties. The leaves were placed in polyethylene trays and subjected to drying in an oven at temperatures of 20, 30, 40, and 50 °C until reaching hygroscopic equilibrium. The experimental data were fitted to several non-linear regression models to characterize the drying process. The Arrhenius model was used to obtain the coefficients of diffusion and the activation energy, which were used to calculate the enthalpy, entropy, and the Gibbs free energy. Midilli and Page were the best models to represent the drying kinetics of bitter melon leaves at temperatures of 20, 30, 40, and 50 °C. Increases in the drying air temperature increased the Gibbs free energy and water diffusivity in the interior of the leaves. Enthalpy and entropy decreased as the temperature was increased.

Introduction
Bitter melon (Momordica charantia L.) is a common vine found in the coastal region and interior of Brazil that is known by its use in cooking and medicine (Joly, 1998). It is a medicinal plant that can be easily obtained; studies report its use in several countries, such as Brazil, China, Colombia, Cuba, Ghana, Haiti, India, Mexico, Malaysia, Nova Zealandia, Nicaragua, Panama, and Peru (Assis et al., 2015).
Drying is one of the main processes that assist in the maintenance of agricultural products, which is needed for a better use and maintenance of the active principle of bitter melon leaves (Rodovalho et al., 2015).
The activation energy represents the difficult level of water molecules to overcome the energy barrier in their migration in the interior of a product (Corrêa et al., 2007). It enables the determination of thermodynamics properties that provide information about the mechanism of control of water vapor sorption (Viganó et al., 2012).
Information on thermodynamic properties in the drying process of leaves is important for designing the drying equipment, calculating the energy required for the process, studying properties of the adsorbed water, evaluating food microstructures, and studying the physical phenomena that occur on the surface of agricultural products (Corrêa et al., 2010).
Therefore, the objective of this study was to evaluate the drying kinetics of bitter melon leaves at temperatures of 20, 30, 40, and 50 °C and determine their thermodynamic properties.

Material and Methods
The Homogeneous leaves without damages caused by pathogens or insects were selected to avoid their effects on the results.
The initial equilibrium water content of the samples was measured after the selection of the product and at the end of the drying by the gravimetric method in a forced-air circulation oven at 103 ± 1 °C for 24 h, with four replications (ASABE, 2010).
The leaves were dried at temperatures of 20, 30, 40 and 50 °C; the temperatures were controlled by a biochemical oxygen demand (BOD) chamber.
The samples were cut using a square template with an area of 17.2 mm 2 , and a thin layer was deposited in polyethylene trays. Approximately 2 g of sample per replication were placed in the trays at the beginning of the drying process, using four replications for each temperature, and were weighed periodically in a digital analytical balance with precision of 0.1 mg until the samples reach equilibrium water content with the drying air.
The interval between weighing started with 20 min and increased up to 10 h to monitor the hygroscopic equilibrium.
The water content ratio was calculated during the drying processes, using Eq. 1.
where: RX -water content ratio, dimensionless; X -leaf water content, kg of water per kg of leaf dry weight; Xe -leaf equilibrium water content, kg of water per kg of leaf dry weight; and, Xi -leaf initial water content, kg of water per kg of leaf dry weight.
The bitter melon leaf water content data were fitted to non-linear regression models (Table 1). Experimental data of drying blackberry (Morus nigra) leaves were also fitted to these models by Martins et al. (2018). Table 1. Non-linear regression models used to estimate the drying of bitter melon leaves RX -Leaf water content ratio (dimensionless); t -Drying time (h); k -Coefficient of drying; a, b, c, and n -Parameters of the models The best models to represent the bitter melon leaf drying kinetics were selected, considering the significance of the coefficients of regression by the t test at p ≤ 0.05, and the magnitude of coefficient of determination (R 2 ), relative mean error (P), and standard deviation of the estimate (SD).
The mathematical model for drying kinetics was selected considering the highest R 2 , p-value below 10%, and the lowest SD.
The standard deviation of the estimate (SD) and relative mean error (P) were calculated using Eqs. 10 and 11.
where: N -number of experimental observations; Y -experimental water content ratio; Ŷ -predicted moisture ratio; and, DF -degrees of freedom of the residue.
The dimensions and mean thickness of the sampled leaves were used to obtain the coefficient of effective diffusion by adjusting the mathematical model of the net diffusion to eight terms, as described in Eq. 12, considering the product geometry as a plain body, disregarding leaf volumetric contractions (Brooker et al., 1992). hp = Planck's constant, 6.626 × 10 -34 J s -1 .

Results and Discussion
The experimental data fitted to the models (Table 2), presenting R 2 above 97%, except the two-term exponential model at temperature of 30 °C (61.71%) and diffusion approach model for temperature of 40 °C (92.97%).
The Midilli model showed the highest R 2 ; high R 2 indicate a better fit of the experimental data to the model (Karizaki, 2016). However, according to Mohapatra & Rao (2005), the use of R 2 as the only evaluation criterion to select drying models is not adequate, thus requiring a joint analysis of fitting indexes.
The Midilli model presented the lowest relative mean error (P) ( Table 2). Mohapatra & Rao (2005) recommend the use of joint analysis of all temperatures involved with the drying process with relative mean error lower than 10%.
The Midilli model presented the lowest standard deviation (SD); according to Draper & Smith (1998), the capacity of a model to describe a physical process with reliability is inverse to the SD, therefore, the lower the SD, the better the fit to the model.
Considering the R 2 , P, and SD, the Midilli model was chosen to represent the drying kinetics of bitter melon leaves.
Studies on drying of other medicinal plant species have used the Midilli model due to its better fit of the experimental data. This was recommended by Corrêa Filho et al. (2018)  The bitter melon leaf drying period decreases as the drying air temperature was increased ( Figure 1); this was because of increases in the pressure gradient between the interior of the leaf and the surrounding air. The water content of bitter melon leaves subjected to air drying at temperatures of 20, 30, 40, and 50 °C was low, approximately 3.20 to 0.35 dry basis. The correlation between increases in the coefficient of effective diffusion (D ef ) and increases in the drying air temperature was evaluated by Eq. 13, which represents the Arrhenius model.
The coefficients of the Arrhenius model were obtained using the logarithm expressed by Eq. 14.
The thermodynamic properties of the dried bitter melon leaves were determined by the method described by Jideanie & Mpotokwana (2009) and Corrêa et al. (2010). Enthalpy (Δh) was calculated using Eq. 15; entropy (Δs) was calculated using Eq. 16; and the Gibbs free energy (ΔG) was calculated using Eq. 17.
Δh -specific enthalpy, J mol -1 ; Δs -specific entropy, J mol -1 K -1 ; ΔG -Gibbs free energy, J mol -1 ; kb -Boltzmann's constant, 1.38 × 10 -23 J K -1 ; and, The drying time decreased as the temperatures was increased. This can be explained by increases in the pressure gradient between the drying air and the interior of the leaves, and indicates a greater decrease of water content in less time.
The Midilli model is also used for the fit of drying kinetics, as recommended by Martins et al. (2018), who evaluated the drying of blackberry leaves at temperatures of 40, 50, 60, and 70 °C.
The k coefficient of the Midilli model increased from 0.077462 to 0.690506 as the temperature was increased (Table  3). This coefficient can be used to characterize the effect of temperature and is related to the effective diffusivity in the drying process in a decreasing period, because the liquid diffusion controls the drying process (Babalis & Belessiotis, 2004).
The D ef values increased in 4.38, 13.9, 24.1, and 38.1 × 10 -10 m 2 s -1 for the drying air temperatures of 20, 30, 40, and 50 °C, respectively (Table 3). Martins et al. (2015) found similar results for Serjania marginata leaves, with a D ef increase from 0.6630 to 12.0712 × 10 -11 m 2 s -1 . According to Goneli et al. (2014a), these increases are dependent on temperature, concentration, vibration of water molecules, and volume of the product. Increase in temperature increases vibration of water molecules in the interior of the product, thus increasing its coefficient of effective diffusion (Goneli et al., 2008).
The D ef of the bitter melon leaf drying was in the range of 10 -11 to 10 -9 m 2 s -1 , which is consistent with Zogzas et al. (1996), who found D ef within this range for agricultural products.
The slope of the Arrhenius curve (Figure 2), generated from the values of ln(D ef ) as a function of the inverse absolute air temperature (1/Ta) during the bitter melon leaf drying, provides the Ea/R ratio, whereas its intersection with the ordinate axis indicates the value of D 0 .
The correlation between the coefficient of effective diffusion and drying air temperature has been described satisfactorily by the Arrhenius equation . According to Gasparin et al. (2017), the linear fit indicates the uniformity of variation of the coefficient of diffusion as a function of temperature.
The activation energy (Ea) obtained by the Arrhenius model was 33.163 kJ mol -1 .  found similar result for Genipa americana leaves, with Ea of 33.87 kJ mol -1 , and Martins et al. (2018) found lower results for blackberry leaves, with Ea of 66.08 kJ mol -1 . The lower Ea found for the bitter melon leaves denotes the need for less energy to trigger the liquid diffusion process than that for blackberry leaves. Different activation energy values found for different agricultural products can be attributed to the physical and biological characteristics of the products .
According to Corrêa et al. (2007), Ea can be explained as the water molecule difficulty to overcome the energy barrier during their migration in the interior of leaves, since the lower the activation energy the higher the water diffusivity in the interior of the leaf during the drying process. The Ea found for the bitter melon leaf drying was within the range (12.7 to 110 kJ mol -1 ) for agricultural products proposed by Zogzas et al. (1996).
Regarding the thermodynamic properties, the enthalpy (∆h) decreased from 30.726 to 30.477 kJ mol -1 as the temperature was increased (Table 4). This indicates the need for a lower quantity of energy for drying at high temperatures .  Table 3. Parameters of the Midilli model (a, b, k, n) and coefficient of effective diffusion (D ef ) for different bitter melon leaf drying conditions  Table 4. Enthalpy (∆h), entropy (∆s), and Gibbs free energy (∆G) of the bitter melon leaf drying process