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Screening of Apiaceae fruits discovered natural resources with considerable biological potential

Abbreviations: TE, Total methanol Extracts; PE, Petroleum Ether fraction; CL, Chloroform fraction; EA, Ethyl Acetate fraction; ME, Methanol residue fraction; BST, Brine Shrimp Test; FRAP, Ferric Reducing Antioxidant Power * Corresponding authors: goodarzi_s@sina.tums.ac.ir doi: 10.29252/jmp.19.76.46 Received 17 September 2019; Received in revised form 22 August 2020; Accepted 25 August 2020 © 2020. Open access. This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (https://creativecommons.org/licenses/by-nc/4.0/) Research Article

Introduction
Cancer is the second global cause of death after cardiovascular diseases. In 2015, 17.5 million cases were reported with cancer which 8.7 million of them have died. Despite the fact that the mortality from cancers was reduced in many countries, it is expected that its incidence will be increased. So, it is estimated by 2030; there will be 26 million new cancer cases and 17 million cancer deaths per year. Ranking of cancer demonstrated breast, lung and other parts of the respiratory system, and colorectal cancers have more incidences in both sexes [1,2].
According to the prevalence and mortality rate of cancer, the importance of the continuing discovery of new anticancer agents is obvious. The potential of natural products and plantderived compounds for cancer prevention and therapy was the reason for increasing attention to them over the previous few years [3]. On the other hand, over 60% of anti-cancer drugs have origins from natural sources or are related to them [4]. A significant portion of currently used antitumor drugs are synthetic or semi-synthetic derivatives of effective constituents elucidated from plants [5].
Apiaceae (Umbelliferae) family contains about 450 genera and 3700 species which can be found worldwide [6]. Iran is an important center of diversification of Apiaceae plants which is represented by 121 genera, 360 species and 122 endemic taxa. The fruits of this family have many culinary and medicinal properties [7,8].
The purpose of present comprehensive study was toxicity investigation of common spices from Apiaceae fruits by brine shrimp lethally test (BST) and MTT assay. In addition, their antioxidant activities and total phenols were determined and compared between different tribes of Apiaceae family to find and introduce active fraction(s) for further studies.

Extraction and fractionation
The fruits were powdered separately and extracted with 80% methanol via maceration at room temperature. Total methanol extracts (TE) were fractionated with petroleum ether (PE), chloroform (CL) and ethyl acetate (EA) and the residue was named methanol (ME) fraction. Total extracts and fractions were kept at the refrigerator prior to the test.

Brine shrimp lethality test (BST)
The toxicity of total extracts and different fractions of fruits of Apiaceae family were determined by brine shrimp test. The eggs of Artemia salina Leach (Shilat Center, Tehran, Iran) were hatched in aerated 35% saltwater under direct light and warmth (28-30 °C eggs transformed to nauplii 48 h later. Total extracts and various fractions of fruits were dissolved in normal saline (1 % (v/v) DMSO was used when necessary) to obtain different concentrations of 1-500 µg/ml in tubes containing 15 nauplii of brine shrimp. The control group contained the vehicle used for dilutions. Dead larvae were counted in each well after 24 h and mortality percentages (p) were determined according to Abbot's formula, p = pi-C/1-C; where pi was the observed mortality rate of each sample and C means the natural larvae mortality of negative control [10]. The lethal concentration (LC50) value of each sample was calculated and reported.

Cell culture and MTT Assay
The colon carcinoma (HT-29), breast adenocarcinoma (MDA-MB-231) and alveolar basal epithelial adenocarcinoma (A549) cell lines were cultured in RPMI 1640 medium and foreskin fibroblast cells (primary culture) was cultured in DMEM medium containing 10 % Fetal bovine serum (FBS) and 1 % penicillinstreptomycin. Cell cultures were maintained at 37 °C in a humidified 5 % CO2 and 95 % air incubator.
Cytotoxicity studies were performed by MTT assay [11]. Growing cells were incubated into 96-well plates at a density of 1×10 4 cells /well. After 24 h, samples with different concentrations (10-200 μg/ml) were added to wells. 48 h later, 20 μl of 5 mg/ml MTT reagent in phosphatebuffered serum (PBS) was added to each well. The plates were incubated at 37° C for 4 h. Then, the medium was replaced by 100 μl pure DMSO to dissolve formazan crystals which were quantified by reading the absorbance at 570 nm on microplate reader (Anthos, Austria). The cell survival was calculated according to the following equation: % Cell viability = Mean absorbance of sample wells/Mean absorbance of control wells × 100 Three independent experiments were performed for each sample. The concentration of samples inducing 50 % growth inhibition (IC50) was obtained from a dose response curve.

Antioxidant power assay
Antioxidant activities of different fractions of Apiaceae fruits were determined by ferric reducing antioxidant power (FRAP) assay [12]. In this method, ferric tripyridyltriazine (Fe (III)-TPTZ) complex was reduced to its blue colored form (Fe (II)-TPTZ) and the absorbance were measured by spectrophotometer. 50 µl of various fractions of fruits with a concentration of 100 µg/ml were added to 1.5 ml of FRAP reagent and incubated at 37 °C. After 10 min, the absorbance of samples was measured at a wavelength of 593 nm. FRAP reagent used as blank and contained 2.5 ml of TPTZ solution (10 mM) in HCl (40 mM), 2.5 ml of FeCl3 (20 mM) and 25 ml of acetate buffer (0.3 M) with pH 3.6. FeSO4. The standard curve was prepared by FeSO4.7 H2O aqueous solution (125-1000 μM) and antioxidant effects of samples were expressed as mM Fe 2+ /100 g of fractions.

Total phenols determination
Total phenols amounts of different fractions of extract from Apiaceae fruits were determined by Folin-Ciocalteu method [13]. By the addition of molybdo tungstophosphoric heteropoly anion (Folin-Ciocalteu) reagent with yellow color to samples, phenol compounds were oxidized and molybdo tungstophophate with blue color was created. The maximum absorption of blue color was achieved in alkaline pH, usually by the addition of NaHCO3 or Na2CO3. 2 ml of Folin-Ciocalteu reagent (1:10 diluted with distilled water) was mixed with 0.2 ml of methanol solutions of Apiaceae fruits fractions (100 µg/ml). After 5 min, 1.5 ml of sodium bicarbonate solution (60 g/L distilled water) was added to the mixture and incubated at room temperature for 90 min. Then the absorbance was measured at 725 nm by spectrophotometer and the experiment was carried out in triplicate. Different concentrations of Gallic acid methanol solution (10-100 mg/ml) were used for the preparation of standard curve and total phenols of samples were reported as gallic acid equivalents (GAE; mg of Gallic acid per g of samples).

Statistical Analysis
The data were average of three samples measurements and reported as Mean ± SD.
Statistical analysis was performed by Graph Pad Prism 5 via One-way ANOVA and post hoc of Tukey, and P < 0.05 were considered as a significant difference.

Brine shrimp lethality test (BST)
Cytotoxic effects of different fractions from Apiaceae fruits were evaluated by BST and MTT assays. Brine shrimp lethality test is a simple, rapid and valid preliminary screening for definition of bioactive cytotoxic chemicals. Mortality ability of all extracts and fractions on Artemia salina were presented in Table 1.
Statistical analysis showed EA fraction of all plants (mean) demonstrated the best mortality rate and had a significant difference with other fractions (P < 0.05) and PE fraction was the second effective fraction (Fig. 1).
Among EA fractions of plants with 10 µg/ml concentration, B. persicum showed the lowest mortality rate with a significant difference with other EA fractions of plants (P < 0.05).
And among PE fractions with 10 µg/ml concentration, D. carota (carrot) is the best and showed significant difference with other PE fractions of plants (P < 0.05) except A. graveolens (dill).   It was considerable greatest cytotoxic fractions against colon carcinoma (HT-29), breast adenocarcinoma (MDA-MB-231) and alveolar basal epithelial adenocarcinoma (A549) cell lines were from Ammineae and Peucedaneae tribes of Apiaceae family.

Antioxidant power assay
Total antioxidant activities of different fractions of Apiaceae fruits were measured based on FeSO4 standard curve (y = 0.001x + 0.049, r 2 = 0.932) and reported in Table 1

Total phenols determination
Total phenols contents of different fractions of Apiaceae fruits were determined according to the standard curve of Gallic acid (y = 0.0076 x, r 2 = 0.999) and reported in Table 1. Some fractions of P. anisum and T. ammi from Ammineae tribe had the highest phenol contents among others. ME and EA were fractions of fruits with higher amounts of phenols except PE fraction of D. carota and P. anisum (after ME) which contained greatest content of phenols. Statistical analysis of total phenols showed no difference between fractions (P<0.05) (Figure 2).

Discussion
The fruits of Apiaceae family are widely used as spices around the world for culinary and medicinal properties [14,15]. One of the important properties of consumption of plants as food is the prevention of chronic diseases including cancers. In the present study, cytotoxicity, antioxidant effects and total phenols of different fractions of twelve fruits from the Apiaceae family were measured and compared in various tribes of Apiaceae family.
The results were demonstrated cytotoxicity of almost all EA fractions and some other fractions of fruits measured by BST method were comparable with well-known cytotoxic alkaloid, berberine hydrochloride with LC50 of 26 µg/ml [16]. In fruits from Ammineae tribe, EA of A. graveolens, PE and EA of B. persicum, EA and ME of P. crispum, EA of P. anisum and EA of T. ammi had significant cytotoxic activities. A previous study about toxicity investigation on A. salina by traditional medicinal plants of Northern Peru showed LC50 of aqueous and ethanol crude extracts of A. graveolens aerial parts as 171 and 25 µg/ml, respectively [17]. There was an opposite report for none lethality activities of different extracts of A. graveolens of Pakistan on brine shrimp [18]. An investigation in Oman demonstrated ethyl acetate and hydro alcoholic extracts of the leaves of P. crispum killed the shrimp larvae with LC50 values equal to 51.95 and 88.15 μg/ml, respectively [19]. Assessment of brine shrimp cytotoxic activity of T. ammi seeds ethanol extract in Sundarbans mangrove forest region revealed LC50 and LC90 as 35.48 and 66.83 µg/ml, respectively [20]. The toxicity of crude extracts of present study was near to mentioned results.
EA of C. sativum from Smyrneae tribe and EA and CL of F. vulgare from Seselineae tribe were effective against A. salina. Aqueous and ethanol extracts of C. sativum aerial parts with LC50 of 22 and 0.015 µg/ml, and F. vulgare with LC50 of >10000 and 2.75 µg/ml exhibited their BST activity [17]. Another study on Sudanese plants demonstrated LC50 of 893.97 and 0.012 µg/ml for F. vulgare aqueous and ethanol extracts, respectively [21]. The data of present work about crude hydroalcoholic extracts of C. sativum and F. vulgare from Iran showed weaker toxicity than alcoholic extracts of Peru and Sudanese plants.
EA, PE and CL of A. graveolens, EA, PE and CL of H. persicum, and PE and EA of F. assa-foetida from Peucedaneae tribe showed potent effects as cytotoxic agents. Cytotoxicity of the aerial part of A. graveolens (dill) crude extract against brine shrimps showed it was toxic with LC50 value of 51.29 μg/ml [22]. A previous report revealed essential oil of H. persicum (LC50 equal to 0.0071 μl/ml) was known as active fraction in BST assay [23]. Our data about toxicity of crude extract of H. persicum was stronger than previous mentioned result.
EA and CL of C. cyminum, and PE and EA of D. carota from Caucalineae tribe were effective via brine shrimp bioassay. There were no previous reports about brine shrimp bioactivities of these plants.
Further cytotoxic investigations of different fractions of Apiaceae fruits were done by MTT assays against three cancer cell lines (HT-29, MDA-MB-231 and A549) and a normal foreskin fibroblast cell line. According to standard of National Cancer Institute (NCI) and Geran protocol, when a crude extract showed an IC50 less than 20 μg/ml, it was highly cytotoxic and active against cancer cell lines, when IC50 equal to 21-200 μg/ml, it is moderately cytotoxic, when IC50 equal to 201-500 μg/ml, it is weakly cytotoxic and when IC50 is upper than 501 μg/ml, it isn't cytotoxic [24]. In details, all fractions showed IC50 higher than 200 µg/ml (weak or non-cytotoxic) except CL of F. assa-foetida on HT-29 with IC50 equal to 144.16 µg/ml; PE and CL of F. assa-foetida on A549 with IC50 equal to 158.03 and 151.94 µg/ml, respectively and CL of A. graveolens (celery) and F. assa-foetida on foreskin fibroblast cell line with IC50 equal to 193.71 and 140.66 µg/ml, respectively.
Based on this criterion, all fractions of fruits demonstrated no significant cytotoxic effects on cancer and normal cell lines. But there were maybe potent cytotoxic compounds in the most cytotoxic fractions of them including P. anisum, F. assa-foetida, H. persicum, A. graveolens (celery), T. ammi and D. carota, or they were maybe active against other cancer cell lines. It was interesting that most of cytotoxic fractions were from Ammineae and Peucedaneae tribes of Apiaceae. There were little reports about cytotoxic evaluation of extracts or fractions of mentioned plants. Antiproliferative effects of A. graveolens seeds extract has been confirmed on Dalton's lymphoma ascites (DLA) and mouse lung fibroblast (L929) cell lines by induction of apoptosis, DNA fragmentation and morphological changes [25]. Another study showed hexane extract of A. graveolens at concentrations of 100 and 200 μg/ml had the best cytotoxic activity on Rhabdomyosarcoma (RD) cell line [26]. In present research, more fractions of Apiaceae fruits were active against breast cancer (MDA-MB-231) cell line including PE of P. anisum and T. ammi from Ammineae tribe with IC50 equal to 147.76 and 195.83 µg/ml, respectively. Previous investigations showed treatment of liver HepG2 cell lines with anise seeds essential oil could exhibited a significant cytotoxicity [27]. In addition, ethanol extract of P. anisum revealed antiproliferative and apoptotic effects toward human prostate cancer cell line (PC-3) with IC50 value of 400 μg/ml [28]. Cytotoxicity of T. ammi essential oil on colon carcinoma cells was confirmed with an IC50 value of 9.6 µg/ml, too [29]. It was shown phytochemicals including thymol, γ-terpinene and p-cymene played an important role in anticancer activity of essential oil and hexane extracts of ajwain [30].
PE of A. graveolens (dill) and CL of H. persicum and F. assa-foetida from Peucedaneae tribe with IC50 equal to 184.36, 177.61 and 155.65 µg/ml, respectively and PE of D. carota from Caucalineae tribe with IC50 equal to 197.00 µg/ml showed cytotoxicity against breast cancer cell line, too. In before experiment, cytotoxicity evaluation of H. persicum aerial parts essential oil on three human cancer cell lines (HeLa, LS180 and Raji) demonstrated no effects with IC50 more than 2 mg/ml [31]. Cytotoxicity of carrot essential oil was reported by IC50 of 35.3 and 46.1 µg/ml on green monkey kidney (VERO) and human pharynx squamous cell carcinoma (FaDu) cell lines, respectively. Carotol, an important constituent of D. carota essential oil exhibited moderate cytotoxicity on both cell lines with no selectivity [32]. In another study, D. carota essential oil induced selective apoptosis in acute myeloid leukemia (AML) cell line via a MAPK-dependent mechanism [33]. It was interesting that most of cytotoxic plants fractions were from Ammineae and Peucedaneae tribes of Apiaceae. previous study showed methanol extract of A. graveolens seeds exhibited better antioxidant effects as Fe +2 chelating, reducing power activities, and the amounts of total phenols were higher in comparison to other extracts [34]. Another research demonstrated P. anisum extract showed the strongest radical scavenging activity among seven Apiaceae fruits from Iran. In addition, ethyl acetate fraction of P. anisum exhibited the highest antioxidant activity and flavonoid content [35]. P. crispum exhibited DPPH free radical-scavenging activity and cupric reducing antioxidant capacity [36]. There were no reports about antioxidant activities of different fractions of mentioned plants.
Total phenol contents of ME of P. anisum, EA of T. ammi and PE of P. anisum from Ammineae tribe were higher than other samples. According to previous results, anethole and thymol were the most abundant components of P. anisum and T. ammi essential oil which have phenol structures [37,38]. It was interesting that all plants with potent antioxidant activities and high phenol contents were from Ammineae tribe of Apiaceae.

Conclusion
The brine shrimp lethally test is an ideal method in the initial biological screening of a broad range of phytochemical compounds including toxic components. Present investigation demonstrated despite potent larvicidal effects of EA fractions from Apiaceae fruits against A. salina, they didn't demonstrate considerable cytotoxicity against cancer and normal cell lines. Statistical analysis also confirmed that EA fraction is the best cytotoxic fraction and there was no significant difference between all fractions from the point of view of total phenols. Almost more cytotoxic fractions belong to fruits from Peucedaneae tribe while all plants with high phenol contents and antioxidant powers were from Ammineae tribe of Apiaceae. Secondary metabolites in active cytotoxic fractions have potential to act as toxic compounds on other cancer cell lines. In addition, there were not seen significant correlations between cytotoxicity, antioxidant and total phenols of different fractions from Apiaceae fruits.


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

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Comparative Analysis of Gross Margin for Three Varieties of Rice Grown under Two Planting Methods and Different Weed Control Measures

Field trials were conducted during the 2005, 2006 and 2007 rainy season at Irrigation Research Station, Kadawa situated in Sudan savannah agro – ecological zone, Kano state, Nigeria to compare the gross margin values obtained from rice production using three different varieties of rice and different weed control measures under two planting methods. The trials were laid out in split – plot design and replicated three times with factorial combination of two planting methods and three varieties in the main plots and weed control treatments in the subplots. The results revealed that transplanting gave better gross margin value with a mean value of 127,053 Naira for the three varieties of rice cultivated under different weed control methods compare to mean value of 113,303 Naira obtained in direct seeded of the same varieties cultivated under different weed control methods. The result of the BCR also showed that transplanting of rice yielded more profit with BCR value of 2.42 for the three varieties of rice cultivated under different weed control methods compared to BCR value of 2.33 for direct seeded rice. FARO 52 performed better among the three varieties of rice with higher mean gross margin values of 143,170 and 120,720 Naira, BCR of 2.60 and 2.42 for transplanting and direct seeded, respectively for the three years cropping season. Application of pre – emergence oxadiazon @ 1.0 Kgha-1 followed by post – emergence piperophos plus propanil @ 1.5 Kgha-1 applied at 5 WAS / T gave the highest gross margin of 166,770 Naira and BCR of 2.78 under transplanting for the three years which was closely followed by hand-pulling weed control measures with gross margin value of 165,720 Naira and BCR of 2.94 under direct seeding compared to the weedy check that gave the least gross margin and BCR in both methods of planting. The study therefore, concludes that transplanting of FARO 52 rice variety and combined pre-emergence application of oxadiazon @ 1.0 kg a.i.ha-1 followed by post-emergence piperophos plus propanil @1.5 kg a.i.ha-1 were the best and second suitable treatment interaction to obtain good profit. Therefore combination is recommended as the economically viable option for lowland rice production in the Sudan savanna agro-ecological zones.

INTRODUCTION
Rice is the world's second most consumed cereal grain [1]. It provides more than one fifth of the calories consumed worldwide by human being. The current national demand for rice in Nigeria is estimated at 5.0 million metric tonnes of milled rice while the current production status is estimated at 3.0 million metric tonnes leaving a deficit of 2.0 million metric tonnes [2]. On analysis, the nutritional value per 100g of milled rice is: carbohydrate-79.95 g, sugars-0.12 g, dietary fibre-1.3 g, fat-0.66 g and protein-7.13 g. It compares favourably with other cereals in amino acid content. Rice contains a low percentage of calcium and its B group vitamins compares favourably to wheat. It also contains 0.07 mg thiamin, 0.04 mg riboflavin, 1.6 mg niacin, 1.01 mg pantothemic acid, 0.16 mg vitamin B 6 , 8 mg Folate, 28 mg calcium, 0.8 mg Iron, 25 mg magnesium, 6 mg phosphorous, 1.0 mg potassium, 1.09 mg zinc, and 1.0 mg manganese [3].
Raw rice may be ground into flour for many uses including, beverages, pudding and bread. The rice bran is high in protein and ideal for use in livestock feed for roughages, protein sources and also used as source of fatty acid. Rice is used in industries mainly for wines, alcoholic beverages, beers, and confectionaries. In Nigeria 'Kunnu' drink is made from raw rice and the popular 'Tuwo' and 'Massa' are also delicacies prepared in Nigeria. The parboiled rice is eaten after boiling with stew or prepared into jollof in homes and during social functions. In fact it is the most popular food at all social functions and meetings irrespective of tribe, religion, or social status. The hulls and husks of grains are used as fuel bedding, incubation materials for making blocks, tiles, fibre board, ceramics, cement and fillers. It is used as a medium for growing mushroom [4], and as fuel in cooking.
Gross margin is the difference between the gross farm income (GI) and the total variable cost (TVC). It is a useful planning tool in situation where fixed capital is negligible portion of the farming enterprises as in the case of small scale subsistence agriculture [5].
Studies on analysis of cost and return of rice production in Ankpa, Nigeria, where results obtained gave the gross margin for rice production as N 43,117.08 ha-1 and benefit cost ratio was 1.70. It was concluded that rice production was profitable in the area, therefore farmers should be encouraged to go into rice production business [6]. Similarly, survey conducted at Kadawa, and Watara indicated that rice production was more profitable amongst small scale farmers compared to medium and large scale farmers. The net return obtained was N 92,697.00 ha -1 by small scale farmers in Kadawa and N 122,547.00 ha -1 by farmers in Watara in the Sudan savanna ecological zone [7]. Madu and Aniobi [8] in their study revealed that the variable cost per hectare for rice production was found to be $360.29 (N 162,130.5) per production cycle, while total revenue of $650 (N292,500.00|) was realized by the respondents. Meanwhile, the gross margin and gross profit were estimated to be $289.71 (N130, 369.50) and $281.56 (N 126,702.00), respectively. The gross profit margin was calculated to be 0.45 which is equivalent to 45%. A study carried out by Oyewole, Akinbola and Ayanrinde [9] in Nassarawa state, Nigeria revealed that rice farmers obtained a mean yield of 4459 Kg of rice per hectare from total variable costs of N212,993.60 Kobo with revenue of N401,310.00 given a gross margin value of N 188,316.40. Toungos [10], reported a gross margin of that ranged between 238, 620 -299,750 Naira, gross return of between 531,240 -543,750 Naira, cost of production of between 244,000 -292,800 Naira and benefit cost ratio that ranged between 1.81 -2.23 when comparing two methods of rice establishment in Mubi North, Adamawa state, Nigeria.
The objective of this study is to compare the gross margin value of three varieties of rice grown under two methods of planting and different weed control measures. Geomorphologically, the Kano region where Kadawa is located is in the Western African plains, with a flat to slightly undulating surface, bordering the Jos Plateau in the Southeast. The prominent weed species of the experimental sites were collected from 1.0 m² areas at random within the plots and the weeds were identified at all sampling stages. The intensities of occurrence were also recorded at the sites of the trials,

Experimental Treatments
The three rice varieties used during the trial were Sipi 692033 (FARO 44), Wita 4 (FARO 52) and ITA 230 (FARO 50). The two planting methods were direct seeding and transplanting, while the seven weed control treatments were Oxadiazon (Ronstar) 25 EC at 1.0 kg a.i.ha  The notable characteristics of these varieties used in this study are given below:

Planting Materials
FARO 44 (Sipi 692033): This variety originated from Asia (Taiwan) and it is cultivated under irrigated swamp condition. It grows to a plant height of 110-120cm (tall variety). It has a tillering capacity of 15-20, with a green stem base. The leaves are long, semi broad and lax. The leaf sheath is green, with fully exerted panicle, erect flag leaf and medium ligule. It matures within 110-120 days, having long grains [11]. The potential yield is 5,000-8,000 kg ha -1 .

FARO 52 (Wita 4):
This variety was developed by the International Institute of Tropical Agriculture (IITA), Nigeria. It is grown as lowland irrigated and shallow swamp rice. It has a plant height of 95-105 cm (medium stature) with tillering capacity of 12-18. The stem base is green with long leaves and green leaves sheath, with a panicle that is fully exerted, and erect flag leaves. The husk from unripe to mature seed is green to straw colour. The stigma is colourless, awnless and the ligule is of medium size. It matures within 125 -130 days and it has short grains [11]. The potential yield is 5,000-8,000 kg ha -1 .

FARO 50 (ITA 230):
This variety was developed by IITA, Nigeria, as irrigated low land swamp rice. It grows to a height of 100-110 cm (tall variety). The stem base is green and the leaves are long with green leaf sheath. The panicle is fully exserted with erect flag leaf. The husk colour form unripe to matured seeds bearing green to straw coloration. The stigma is colourless, it is awnless and ligule is medium. It matures in 120 -125 days and produces short grains [11]. The potential yield is 5,000-8,000 kg ha -1 . Paddy yield (kg/ha): Harvested paddy from each net plot was threshed after sun drying, winnowed and the weight obtained expressed on per hectare basis and recorded accordingly.

Gross Margin Analysis
Gross margin was done to determine profitability of rice production with the following model: Where, GM= Gross margin TR=Total revenue TVC=Total variable costs.
Fixed cost arising from simple tools such as hoes, cutlasses and sickles are negligible as such can be ignored when calculating profitability of small scale rice production [12].

Operating Cost Ratio (OCR)
The operating cost ratio was estimated for the three varieties of rice considering the two methods of planting and weed control measures adopted. The OCR is calculated by dividing the total variable costs by the total returns. This established the proportion of the gross income that goes to service the operating expense of the respondents and this is directly related to the farm variable input usage. As a rule, an operating ratio of one means that the gross income just defray the expenses incurred on the variable inputs used on the farm. The formula is given below: Operating Cost Ratio = Total Variable Costs / Total Returns.

Benefit Cost Ratio (BCR)
This was estimated for the three varieties of rice considering the two methods of planting and weed control measures adopted. The BCR is calculated dividing gross return by the total variable cost. BCR indicates safety in business. Higher BCR indicates safer the business. The formula is given below: Benefit Cost Ratio = Total Returns / Total Variable Costs

Gross margin analysis estimates
Average cost and returns per hectare for lowland rice varieties under transplanting and weed control method studied in the combined mean of 2005-2007 is contained in Table 1 and that of direct seeding method in Table 3.
The data in Table 1 (Table 1). Similarly, highest gross margin value of N 166,770.00 was also obtained from the combined application of oxadiazon followed by piperophos plus propanil compared with N 38,501.00 ha -1 obtained with the weedy check.
Similarly, Table 3 shows that FARO 52 gave the highest gross margin of N 120,770.00, followed by FARO 44 with N 113,570.00 while the least value of N 105,570.00 was obtained from FARO 50.
Similarly, using the mean value for the three varieties of rice to estimate the gross margin under different weed control measures for direct seeding showed that hand -pulling method produced the highest returns of N 250,950.00 ha -1 compared to the least of N 121,600.00 obtained from the weedy check (Table 3). The highest gross margin value of N 165,720.00 ha-1 was also obtained from the hand -pulling method compared with N 48,370.00 ha-1 obtained with the weedy check ( Table 3).
Comparison of the two methods of planting showed that FARO 52 was best variety that gave both highest return and gross margin values under both transplanting and direct seeding. However the transplanting method recorded a higher return and gross margin values than the , respectively were recorded under transplanted method along with pre-emergence application of oxadiazon @ 1.0 kg a.i.ha -1 . It was followed by post emergence application of piperophos plus propanil @ 1.5 kg a.i.ha -1 which was higher compared to the values of N 250,950.00 ha -1 and N 165,720.00 ha-1 obtained with handpulling method in direct seeding. The mean value for transplanting for the three varieties of rice for return and gross margin were also higher than those obtained in direct seeding.  Table 2, it revealed that weedy check had the highest value of 0.67 compared to the rest treatments. However, lowest value of 0.36 was obtained for piperophos plus propanil alone and oxadiazon followed by, piperophos followed by, propanil at 5WAS /T, respectively. This was closely followed by handpulling method with an OCR value of 0.37.

Gross margin measures profitably and estimated revenue, less total variable cost
This showed that more of the returns using weed check method goes to defray the operating costs in rice production compare to the other weed control measures.  Table 4 also revealed that weedy check had the highest value of 0.60 compared to the rest treatments. However, lowest value of 0.34 was obtained for hand-pulling method. This showed that more of the returns using weed check method goes to defray the operating costs in rice production compare to the other weed control measures.
Comparison of the two methods of plating clearly showed that in term of rice varieties, the OCR of direct seeding mean value 0.43 is higher than that of 0.41 obtained for transplanting. This showed that more gross margin is obtained using transplanting method than the direct seeding. More return from direct seeding goes to defray the operating costs compare to transplanting. Similarly, the result revealed that transplanting method performed better with a mean OCR (0.41) compared to 0.43 OCR for direct seeded under different methods of weed control.

Benefit Cost Ratio (BCR)
The result of benefit cost ratio considering rice varieties, weed control methods under transplanting and direct seeding methods are presented in Table 2 and Table 4, respectively. The result in Table 2 (2.81). This was closely followed by the application of oxadiazon followed by, piperophos followed by, propanil at 5WAS /T with BCR (2.78). The result revealed that if weeds are left on the rice farm without control, the profitability of the rice production will declined. So weed control measures help to increase output of rice.
The result in Table 4  Comparison of the two methods of planting/sowing clearly showed that in term of rice varieties the BCR of direct seeding with mean value 2.33 is lower than that of 2.42 for transplanting., It showed that more gross margin is obtained using transplanting method for rice production than the direct seeded. Similarly, the result revealed that in term of using different methods of weed control for rice production transplanting method also performed better with mean BCR of 2.42 compared to that of direct seeded BCR of 2.33.

Discussion
The result revealed that good returns with good gross margins were made from cultivating rice in the study area using different rice varieties and different weed control measures under the two planting methods. When comparing the two methods, transplanting gave higher returns and gross margins compared to direct seeding. This may be as a result that direct seeding may witness poor germination rate compare to establishment of transplanted rice which may result into low plant population and low output. The optimum returns and gross margin values obtained in this study was in agreement with different studies. Oyewole, Akinbola and Ayanrinde [9], reported a gross return of N401310.00 and gross margin value of N 188,316.40 in their study. Toungos [10], reported a gross margin of 238, 620 Naira for system of rice intensification, 299,750 Naira for traditional method of rice production in Mubi North, Adamawa state, Nigeria.
The BCR values obtained for both method of planting using different varieties of rice and different methods of weed control showed that rice production in the study area for the three cropping years was profitable and it is a business that is safe to invest on. The BCR values obtained in this study were higher than the BCR of 1.81 reported by [6] but similar to the range of 1.93 -2.44 reported by Ali et.al.
[13] in their study titled comparison of different methods of rice establishment and nitrogen management strategies for lowland rice.

CONCLUSSION
Based on the result of this study, transplanting of FARO 52 combined with pre-emergence application of oxadiazon @ 1.0 kg a.i.ha -1 followed by post-emergence piperophos + propanil @ 1.5 kg a.i.ha -1 gave highest gross margin of N 166,770.00 from the recommended combined herbicide compared to N 38,501.00 ha -1 in the unwedded control, N 127,053.00 with transplanting FARO 52 compared to N 113,303.00 in direct seeding. Therefore, it can be concluded that transplanting of FARO 52 rice variety and combined application of preemergence application of oxadiazon @ 1.0 kg a.i.ha -1 followed by post-emergence piperophos plus propanil @ 1.5 kg a.i.ha -1 could hereby be recommended as economically viable integrated package for lowland rice production in the sudan savanna agro-ecological zones.


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Post-harvest Practices of Maize Farmers in Selected Counties in Kenya and Isolation of Mycotoxin Producing Fungi from Maize Samples

Mycotoxins produced by fungi are among the leading courses of human mortality and morbidity all over the world. This study aimed at establishing the post-harvest practices that lead to contamination of maize with fungi in Trans Nzoia, Kisii, Kisumu, Bungoma, Migori, Kericho, Machakos, Kitui and Meru Counties in Kenya. The study was also aimed at isolating the most common mycotoxin producing fungi from maize samples collected from the study areas. The post-harvest practices were determined using a questionnaire. From each county, a total of 130 maize samples were collected. The samples from each county were mixed to make a composite sample. The moisture content of the maize samples were determined and percentage moisture content calculated. Each sample was ground using a dry mill kitchen blender (BL335, Kenwood, UK). A sample of 1g was suspended in 9mL of sterile distilled water and serial dilution up to 10 -2 carried out. A sample of 0.01mL was plated on potato dextrose agar. Mycotoxin producing fungi were identified using morphological characteristics and fungal identification keys. The postharvest practices within the counties varied significantly. The mean moisture content of stored maize varied from 16.78% in Kitui to 19.33% in Trans Nzoia. The number of mycotoxin producing fungi ranged from 10.33 CFU/g in Machakos to 61.00CFU/g in Trans Nzoia. This study established that the post-harvest practices observed in the study areas led to contamination of maize with mycroflora. In addition, the moisture content levels of stored maize in the counties favoured growth of mycotoxin producing fungi. There is need to identify the mycotoxins produced by the mycotoxin producing fungi isolated in this study.

INTRODUCTION
The term mycotoxin was coined in 1960 to refer to toxins basically found in contaminated pea nuts in animal feed and the loss of turkeys in England (Cruz et al., 2013). The mycotoxin was there after identified as aflatoxin B1 produced by Aspergillus flavus (Pascual et al., 2016).
Mycotoxins are natural products produced by fungi that evoke a toxic response when introduced in low concentrations to higher vertebrates and other animals by a natural route (Hussien et al., 2017). In addition, mycotoxins are secondary metabolites that act as metabolic intermediates. They occur as differentiation products in restricted taxonomic groups. Mycotoxins are biosynthesized from one or more general metabolites by a wider variety of pathways than is available in general metabolism (Mathana et al., 2017). The term mycotoxin was later applied to other toxic fungal natural products (Makuvaro et al., 2014).
Early taxonomist categorized mycotoxins into two groups. Those that invade seed crops were categorized as field fungi and they included Cladosporium sp., Fusarium sp. and Alternaria sp. These fungi gain access to seeds during plant development stage (Maina et al., 2015). The second category were the storage fungi such as Aspergillus sp. and Penicillium sp, which infect the stored produce during storage (Baquião et al., 2013). However, this categorization is not strictly being followed (Turner et al., 2015). Hussien et al. (2017), categorized fungi into four major groups. The first group formed the plant pathogens such as Fusarium graminearum and Alternaria alternate while the second group was composed of fungi that grow and produce mycotoxins on aged or stressed plants viz F. moniliforme and Aspergillus flavus. Fungi that initially colonize the plant and increase the feedstock's susceptibility to contamination after harvesting such as A. flavus were placed in the third category while those fungi that are found on the soil, decaying plant material, developing kernels in the field constituted the fourth group. These fungi proliferate later in the store if conditions allow and they include P. verrucosum and A. ochraceus.
Basically, most of the mycotoxin producing fungi are ubiquitous in soil and other substrates (Omara et al., 2020). The mycotoxins cause many diseases to crops, insects and animals including humans. Fungi infect agricultural crops including cereals and a variety of oilseeds (Wacoo et al., 2019). Seetha et al. (2017) maintained that production of mycotoxins is species specific. This demands for proper identification and characterization of fungi in order to develop any prevention strategy. A wide variety of mycotoxins have been documented as contaminants of poultry feed, most important of which are aflatoxins (B1, B2, G1, G2) and Ochratoxin A (OTA) (Marin et al., 2012). Aflatoxins are the most studied group of mycotoxins (Czembor et al., 2015). Apart from producing clinical toxicosis, aflatoxins also reduce resistance to diseases and interfere with vaccine induced immunity in poultry (Amra et al., 2017). Pascual et al. (2016) advocates for regular monitoring of toxigenic mycoflora of the agricultural based feeds and foods as an essential pre-requisite for the control of mycotoxicosis among animals and humans. The highest occurring mycotoxins in nature are aflatoxins which are produced mainly by A. flavus and A. parasiticus (Gong et al., 2016). At least 16 structurally close aflatoxin has been detected to date. However, the most common are aflatoxins B1, B2, G1 and G2. (Antonio et al., 2018). The most toxic causing hepatocarcinogenic among other ailments (Algabr et al., 2018).
The world has witnessed high levels of crop and livestock losses resulting from contamination of toxigenic fungi and mycotoxins (Williams et al., 2014). Economic loss have increased enactment of regulations that control conducting regulatory programs to prevent such contamination. Feeding livestock with aflatoxin contaminated feeds may lead to death, immune suppression, growth reduction and reduced yields in both animals and crops (Mboya et al., 2011). Besides, intake of aflatoxins contaminated animal and crop produce jeopardizes human existence (Czembor et al., 2015). The major aims of this study were to identify the maize post-harvest practices in Trans Nzoia, Kisii, Kisumu, Bungoma, Migori, Kericho, Machakos, Kitui and Meru counties and to isolate mycotoxin producing fungi from maize samples collected from the study areas.

MATERIALS AND METHODS The study areas
The study was carried out in different geographical locations of Trans Nzoia, Kisii, Kisumu, Bungoma, Migori, Kericho, Machakos, Kitui and Meru Counties. The counties had recently reported cases of mycotoxicosis (Omara et al., 2020). Trans-Nzoia County is an agricultural county situated in Western Kenya at an altitude of 2,100 m above sea level. Temperatures in this region range from a low of 9.05 °C to a high of 26.85 °C. The average rainfall ranges from 950 mm to 1500 mm annually (Gachara et al., 2018).
Kisii county is located on Latitude: 0° 41' 0 N and Longitude: 34° 46' 0 E. The county has a short rainfall season, between September and November and long one between February and June. The annual rainfall is over 1,500 mm per annum of rainfall with temperatures ranging from 16 o C to 27 o C. The County has a population of 1,152,282 (Sichangi et al., 2020).
Kisumu County has a population of 1,155,574. The county covers an area of 2085.9 km 2 and an annual relief rainfall that ranges between 1200 mm and 1300 mm. Kisumu County lies within longitudes 33° 20'E and 35° 20'E and latitudes 0°20'South and 0°50'South (Juma et al., 2018). Bungoma County has a population of 1,670,570 and an area of 2,069 km 2 . The economic activities of Bungoma County are mainly agricultural, centering on the sugarcane and maize industries. The area experiences high rainfall throughout the year, and is home to several large rivers, which are used for small-scale irrigation. The county is located at 00°34′00″N 34°34′00″E (Odhiambo et al., 2013).
Migori County is located in Western Kenya and has a population of 1,116,436 persons. The County has two main rainy seasons. The first rainy seasons starts in March and ends in May and is called long rains. The second season called "opon" starts in September and ends in November. The minimum average temperature is 24 o C and the maximum 31 o C (Islam et al., 2018). Kericho County is located between longitude 35 o 02' and 35 o 40' East and between the equator and latitude 0 23' South with an altitude of about 2002m above the sea level. It has a population of 901,777 (2019 census) and an area of 2,111 km 2 . The County has a temperature range and a population density 370 people per Km 2 (Rotich et al., 2017).
Machakos County has a population of 1,421,932. The county is located at latitudes 0º 45' S to 1º 31' S and longitudes 36° 45' E to 37° 45' E. It has a semi-arid climate with hilly terrain and stands at an altitude of 1000 to 2100 m above sea level. Residents of this County practice subsistence agriculture farming maize and drought-resistant crops such as sorghum and millet. The county covers an area of 6,208Km 2 . The County lies at altitude of 1000-1600 meters above sea level (Islam et al., 2018). Kitui County is situated in Eastern Kenya. It is located at co-ordinates of 1° 22' 0" South and 38° 1' 0" East. The County has a population of 1,012,709 people and an area of 20,402 Km 2 . The average rainfall in this area ranges from 500 mm to 1050 mm per annum. Long rains occur between March and May while short rains are experienced during October and December. Temperatures are normally high with the mean ranging from 26 °C to 34 °C. Residents of Kitui County practice subsistence farming as their main source of livelihood (Gachara et al., 2018).
Meru County is located in Eastern Kenya with a population of 987,653 and a population density of 6 people per Km 2 . The County is located at 0.047035 o N and 37.649803 o E. The County experience an average temperature range of 8 o C-32 o C. The rainfall ranges between 300 mm and 2600mm per annum. The long rains come in April and May and short rains in November and December. The hot months are in the Months of June, September, January and February. The main sources of livelihood of the residents of Meru County are agriculture and trade .

Administration of questionnaires
Questionnaires were used to collect data on post-harvest practices practiced in the study areas (Appendix I). The respondents were asked questions regarding source of maize, stage of maize harvesting, use of poor grade maize, maize vendor types and storage methods of maize. The collected responses was used to make conclusions based on the experimental results (Czembor et al., 2015).

Sample collection
Maize grains samples were collected from the various storage structures of farmers from Trans Nzoia, Kisii, Kisumu, Bungoma, Migori, Kericho, Machakos, Kitui and Meru Counties. The formula below was used to calculate the sample size at 95% confidence level and a desired level of precision at 5% (Algabr et al., 2018).
Where n=sample size, Z= confidence level at 95% (1.96), p= estimated proportion of the sample population, q= (1-p) and e= desired level of precision at 5% with a standard value of 0.05. Based on this formula at 5% precision and a Z value of 1.96, the sample size was determined as; The samples from each County were then mixed to form a composite sample. The samples were packed in new khaki bags and transported to Egerton University, Department of Biological Sciences laboratories. The samples were stored at 4 o C awaiting mycological analysis.

Determination of moisture content (MC)
Moisture content of each sample was determined by drying 5 g of the sample for 2 hours at 105 o C in a hot air oven (ADP21/31 Yamato Scientific, America). The samples were allowed to cool at room temperature (20±2 o C). The dry weight was recorded and used to calculate the moisture content expressed as a percentage using the formula below (Domenico et al., 2016); Where: M 0 was the initial weight of the maize sample and M 1 was the final weigh of the maize sample. Isolation of mycotoxins producing mycoflora Surface sterilization of maize kernels was carried out using 70% ethanol for 2 minutes and rinsed twice in sterile distilled water to remove external contaminants (Nyongesa et al., 2015). The maize kernels were dried using sterile Whatman filter papers. One kilogram of each maize sample was ground using a dry mill kitchen blender (BL335, Kenwood, UK). One gram of each ground maize sample was suspended in 9 mL of sterile distilled water. The mixture was thoroughly shaken and serially diluted up to 10 -2 . A sample of 0.01mL was plated on sterile Potato Dextrose Agar (PDA) media and incubated at 28 o C for 7d (Wacoo et al., 2014). The isolates were subcultured on PDA to obtain pure cultures. The fungal isolates were enumerated as colony forming units per gram of maize (CFU/g) as follows ;

Identification of the mycotoxin producing mycoflora
The isolated fungal species were identified morphologically using their macroscopic and microscopic characteristics (Nyongesa et al., 2015). Macroscopic features that were observed included colour of the colony, size of the spores, their texture and pattern. Microscopic features used in identifying the isolates were elevation of the philiades, the size of the conidiohores and protrusion of the hyphae (Lee et al., 2013). Fungal identification keys were also used to further characterize the isolates (Mathana et al., 2017).

Data analysis
Analysis of variance was carried out using PROC ANOVA procedure of Genstat Discovery 2 statistical software (Lawes Agricultural Trust, Rothamsted Experimental Station 2006, version 9). The mean differences were compared using the Fisher's protected least significant difference test at 5% significance level.

Post-harvest practices of maize farmers
The source of maize was evaluated since it affected the mycoflora recovered from maize. A proportion of 77.3% obtained their maize from the family farm (Table 1). This was the most common source of maize within the study areas. However, the least common source of maize were the millers (0.95). The sources of maize varied significantly (F=0.55 P=0.0081).
People within the study area harvested maize at different stages. Most people (93.9%) stacked maize and give it time to dry (Table   1). A minority of the farmers (1.1%) harvested and sold their maize when green. Stage of harvesting maize varied significantly within the study areas (F=8.78 P=0.001).
Farmers in the study areas used poor grade maize in different ways. Some farmers (94.4%) preferred feeding livestock with the poor grade maize. However, 1.1% of the farmers threw the maize away while others used it at home. The utilization of poor grade maize differed significantly from each other (F=5.2 P=0.001).
The type of maize vendor determines the level of contamination of maize. Farmers in the study areas obtained maize from different sources. Most of the farmers (92.5%) obtained maize from grain stockist while 1.1% sourced maize from migrant venders. The sources of maize were significantly different within the study areas (F= 0.19 P=0.0098).
Different farmers stored maize differently after harvesting (Figure 1). Storing maize in sisal bags was the most common method (44.6%). However, a minority (1.3%) stored their maize in polythene bags. Maize storage methods differed significantly within the study areas (F=0.13 P=0.009).

Number of the most common mycotoxin producing fungi isolated from maize samples
The Aspergillus sp. ranged from 11 CFU/g in Kitui to 63 CFU/g in Trans Nzoia, Penicillium sp. from 13 CFU/g in Kitui to 62 CFU/g in Kisii and Fusarium sp. from 9 CFU/g in Machakos to 59 CFU/g in Trans Nzoia (Table  3). The mean mycoflora isolates varied from 10.33 CFU/g in Machakos to 61.00 CFU/g in Trans Nzoia. There was no significant difference in the mean MC among the study areas (F=0.08 P=0.40). Means within a column followed by the same superscripts are not significantly different at P≤0.05. The P-values were calculated using SPSS P-significant tests. (Nyongesa et al., 2015). It offers most Kenyan communities with food security (Wagacha et al., 2013). As a result, it's produced in many places in the country. However, maize production is threatened by prevalence of a wide variety of fungi some of which produce mycotoxins that are toxic to humans.

Maize is the staple food in Kenya
According to Akowuah et al. (2015), maize source greatly influences the levels of maize contamination with fungal spores. In this study, most people obtained maize from family farms while the minority got them from millers. This agreed with a previous study by Williams et al. (2014). This may be attributed to similarity in the regions from which the samples were obtained.
Stage of harvesting maize influences growth of fungi on the produce (Mboya et al., 2011). Majority of the respondents in this study stalked maize in heaps and allowed them to dry while some of them harvested and sold the maize at green stage. These results differed with those of a previous study carried out by Voss et al. (2014). Variations in the respondent's economic status could have contributed to the differences (Queiroz et al., 2012). Makuvaro et al. (2014) maintained that farmers with a good economic status delayed harvesting of maize thus allowing it to dry properly before harvesting. This reduces the chances of fungal growth on the harvested maize (Amra et al., 2017). Farmers living in areas that receive high amounts of rainfall obtain many poor grade maize due to fungal infection before harvesting than those in dry areas (Antonio et al., 2018). The poor grade maize is used in a variety of ways (Lee et al., 2013). In the present study, most farmers fed livestock with the poor grade maize. However, 1.1% farmers threw the poor grade maize away. In addition, 1.1% of the farmers used the maize at home. These results disagreed with those of a previous study by Bii et al. (2012). This may be attributed to differences in the levels of knowledge about dangers of using poor grade maize . Feeding animals with poor grade maize is unsafe since mycotoxins produced by fungi present in the maize may end up in the animal products such as milk (Wacoo et al., 2019). The animal products are unsafe for human consumption (Soares et al., 2012).
The study areas had different maize vendors such as grain stockist, migrant vendors, posho millers and commercial millers. Majority of the respondents in the study areas obtained maize from maize grain stockist while the minority sourced maize from migrant vendors. These results contradicted those of a study carried out on occurrences and frequency of fungi and detection of mycotoxins on poultry rations in Yemen by Algabr et al. (2018). This could be attributed to differences in the ability of the vendors to reach the respondents (Viebrants et al., 2016). According to Czembor et al. (2015), the ability of maize vendors to transport maize affects the accessibility of the produce to potential buyers. In addition, the type of maize vendors affects the contamination level of maize by mycroflora. This result from differences in maize handling practices among vendors (Perrone et al., 2014).
Storage method affects moisture content in maize ( Baranyi et al., 2013). High moisture content favours growth of mycoflora leading to increased production of mycotoxins (Warth et al., 2012). In the present study, 44.6% of farmers stored maize in sisal bags while 1.3% stored maize in polythene bags. These results differed with those of a previous study by Seetha et al. (2017). This could be attributed to differences in knowledge about the right containers for maize storage among respondents. In addition variations in the ability to buy the right maize storage container may be a contributing factor (Gong et al., 2016).
Fungi require a certain water activity for optimal growth (Omara et al., 2020). High moisture content levels were found in the majority of the samples in this study. Similar results were obtained by Brandyopadhay et al. (2016) when carrying out a study on biological control of aflatoxins in Africa. This could be attributed to similarity in agro ecological zones from which the samples were collected. The recommended moisture content in maize and other cereals is 13% (Wacoo et al., 2014). Trans Nzoia recorded the highest mean moisture content (19.33%) while Kitui recorded the least (16.78%). The high moisture contents observed in the current study may be attributed to the rising cases of mycotoxin contamination in the study areas. Previous studies show that fungi grow optimally at moisture contents above 15% and low or no growth at moisture contents of or below 12% (Palmer et al., 2013). This indicated that most of the maize samples in the present study were at risk of mycoflora attack (Ehrlich & Mack, 2014).
Aspergillus sp., Penicillium sp. and Fusarium sp. were the main mycotoxin producing fungi isolated in this study. This agreed with a previous study by Marin et al. (2012). This could be attributed to similarity in the study areas in terms of environmental conditions (Domenico et al., 2016). Ismaiel and Papenbrock, (2015) explained that the fungal isolates differ depending on the agro ecological zones. The highest fungal isolates were obtained from Trans Nzoia while the least were obtained from Machakos. Trans Nzoia is a humid region while Machakos is a semi-arid area with low humidity levels a factor that may have led to the observed results. In addition, variations in the number of fungal isolates within the study areas could be attributed to differences in maize management practices after harvesting (Cruz et al., 2013).

CONCLUSION
The maize post-harvest practices observed in the study areas led to contamination of maize with mycoflora. The moisture content levels of stored maize favoured growth of mycotoxin producing fungi.

Recommendations
There is need to identify the species of the mycotoxin producing fungi obtained in this study. The mycotoxins produced by the fungal species needs to be established.


extfieldsofstudy: Medicine Biology

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Pathogenicity and Molecular Typing of Fowl Adenovirus-Associated With Hepatitis/Hydropericardium Syndrome in Central China (2015–2018)

In central China, a large number of broiler and layer flocks have suffered from outbreaks of severe hepatitis/hydropericardium syndrome (HHS). This resulted in huge economic losses to the poultry industry, from 2015 to 2018. To identify the specific pathogen and study its pathogenicity, 195 samples from Hubei, Jiangxi, Anhui, Hunan, and Henan provinces in central China were collected. The samples were screened for the adenovirus hexon gene, and neighbor joining was used for the phylogenetic reconstruction of the sequences. Among the collected samples, 122 were found to be positive for fowl adenovirus (FAdV) by PCR, and 73 isolates were obtained. The predominant viral serotype was serotype 4 (FAdV-4), which was found in 48 isolates, while 24 were serotype 10 (FAdV-10), and one was serotype 2 (FAdV-2). The CH/HBTF /1710 isolate was selected for further experiment and inoculated into 33-day-old specific pathogen-free chickens via intramuscular injection or oral administration to evaluate pathogenicity. It was found that the mortality for chickens infected by intramuscular injection or oral administration was 70 and 60%, respectively. Necropsy revealed mild to severe hepatitis and hydropericardium at 5 and 7 days after infection. Ancestor analyses indicated that all of the FAdV-4 strains obtained in this study shared a common Indian precursor and had a close genetic relationship with the JSJ13, SDSX, HN/151025, and SDDM-15 strains common in China.

INTRODUCTION
Fowl adenoviruses (FAdVs) are non-enveloped viruses containing double-stranded linear DNA genomes and belonging to the genus aviadenovirus. FAdVs are grouped into five species (FAdV-A to FAdV-E) with 12 serotypes (FAdV-1 to −8a and −8b to −11) based on restriction enzyme digest patterns and serum cross-neutralization tests (1). The most notable diseases associated with FAdVs infection in chicken are the inclusion body hepatitis (IBH), hepatitis/hydropericardium syndrome (HHS), and gizzard erosions (GE) (2)(3)(4). All 12 serotypes of FAdVs have been associated with outbreaks of IBH, whereas HHS is primarily caused by FAdV serotype 4 (FAdV-4) strains, which has been classified as FAdV-C (5). IBH is mainly observed in 3-to 5-week-old broiler chickens, and the mortality approaches 10% (6). The main lesions of IBH are hepatic necrosis with microscopic eosinophilic or basophilic intranuclear inclusion bodies in hepatocytes (7,8). HHS, also called hydropericardium syndrome (HPS), is an emergent, immunosuppressive disease of 3-to 6-week-old broilers, characterized by sudden onset and mortality from 30 to 70%, accumulation of clear or amber-colored liquid with aqueous or gelatinous consistency in the pericardial sac, enlarged, mottled, and friable liver with infiltration of mononuclear cells, and presence of basophilic intranuclear inclusions in hepatocytes (9)(10)(11).
HHS was first reported in Angara Goth, Pakistan, in 1987 and subsequent outbreaks have been recorded in many other countries, resulting in significant economic losses to poultry raisers in the United States, Canada, Germany, and Korea (5,7,10,12). From 2015, severe HHS induced by FAdV-4 has emerged across several different areas in China, and clinical cases of HHS have been increasing since 2016 (11,13). From 2015 to 2018, many cases of suspected FAdV infection occurred among broilers and layers in central China. Most of the cases showed severe HHS and caused high mortality resulting in tremendous economic losses to poultry farmers. The objective of the present study was to carry out the molecular typing of FAdV strains from HHS cases in Hunan, Hubei, Anhui, Jiangxi, and Henan provinces of central China and to study the pathogenicity of these strains in specific pathogen-free (SPF) chickens.

Eggs and Chickens
Specific pathogen-free (SPF) white nick chickens and embryonated eggs were obtained from Beijing Merial Vital Laboratory Animal Technology Co., Ltd (Beijing, China). The birds were maintained in isolators under negative pressure with ad libitum feed and water.

Sample Collection and Treatment
From 2015 to 2018 in central China, liver samples were collected from broiler and layer chickens suspected of having HHS, characterized by pericardial effusion or hemorrhagic hepatitis. The tissue samples were homogenized in phosphate-buffered saline (PBS; 0.1 M, pH 7.2) at a ratio of 1:5. After three freezethaw cycles, the homogenates were centrifuged at 8,000 g for 20 min at 4 • C. The supernatants were removed, passed through a 0.2-µm filter and immediately stored at −70 • C until PCR analysis, and virus isolation was performed (11).

FAdV Detection by PCR and Sequencing
Total DNA was extracted from supernatants using a DNA extraction kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The adenovirus hexon gene was used to study the taxonomy and antigenic properties of FAdVs (14). Based on the highly conserved region of the hexon gene, specific primers H1 (H1f, 5 ′ -TGGACATGGGGGCGACCTA-3 ′ , H1r, 5 ′ -AAGGGATTGACGTTGTCCA-3 ′ ) and H2 (H2f, 5 ′ -AACGTCAATC CCTTCAACCACC-3 ′ , H2r, 5 ′ -TTGCCTGTGGCGAAAGGCG-3 ′ ) were designed to amplify the complete coding sequence of the hexon gene of FAdV. PCRs were performed with the following parameters: 94 • C for 5 min followed by 30 cycles of denaturation at 94 • C for 1 min, annealing at 55 • C for 1 min, and extension at 72 • C for 1 min with a final extension at 72 • C for 10 min. The PCR products were run on a 1.0% agarose gel. The reaction volume was 25 µl consisting of 1 µl (10 pmol) of each primer, 12.5 µl of Taq SuperMix (TaKaRa, Dalian, China), 2.5 µl of DNA, and 8 µl of nuclease-free water. The amplified products were cloned into the pMD18-T vector (TaKaRa, Dlian, China) for sequencing. After sequencing, the hexon genes were assembled using the Seqman program in the DNASTAR software package.

Phylogenetic Analysis of the FAdV hexon Gene
The nucleotide sequences of the hexon genes were aligned with homologous sequences using the Lasergene sequence analysis software package (DNASTAR Inc., Madison, WI, USA) and were compared to reference sequences using MegAlign. The reference isolates included strains from the five species (FAdV A-E) with 12 serotypes (FAdV-1 through 7, 8a, 8b, and 9-11). A phylogenetic tree was constructed using the neighbor-joining method of MEGA version 5.2, and bootstrap p-values were determined from 1,000 replicates of the original data (15).

Viruses Used for Experimental Infections
Samples that were positive by PCR were inoculated into monolayer cultures of 15-day-old SPF chicken embryo liver cells (CELC). Cultures of 5 × 10 5 cells/dish (60-mm culture plates) were incubated at 37 • C in 5% CO 2 . Aliquots of 0.2 ml of supernatant were pipetted onto CELC, and virus adsorption was allowed to take place at 37 • C for 2 h. The culture supernatants were then discarded, and fresh Dulbecco's modified Eagle's medium supplemented with 2% fetal bovine serum was added to the CELC. Supernatants and cells were harvested after 48h incubation, and three blind passages were performed. The determination of the median tissue culture infectious dose (TCID 50 ) of the FAdV isolates in CELC was conducted according to a standard procedure, and TCID 50 values ranged from 10 4.75 to 10 7.5 . For infection, the CH/HBTF/1710 isolate was used at 10 7.5 /0.1 ml.

Pathogenicity of FAdV Isolates in SPF Chickens
Seventy 33-day-old SPF white nick chickens were used in this study. The chickens were randomly divided into three groups with 20 birds per group. One group was infected with the CH/HBTF/1710 isolate by intramuscular injection, a second group was infected via the oral route, and the remaining 20 chickens were used as uninfected controls. The infectious dose was 10 7.5 TCID 50 per chicken in a volume of 0.2 ml, and the control chickens received the same volume of sterile PBS. In addition, 10 chickens were marked as contact sentinels and added to the oral route group given PBS only. The chickens were housed in isolators under negative pressure, observed twice a day and observed for clinical signs for 21 days. Clinical signs were scored as follows: 0 = normal, 1 = mild depression, 2 = severe depression, 3 = paralysis/prostration, and 4 = death (16). Dead chickens were necropsied to assess gross pathologic lesions, and samples of heart, liver, spleen, thymus, and kidney tissues were fixed in 10% neutral-buffered formalin for histology analysis.

Histopathology and Immunohistochemistry
Samples of heart, liver, kidney, spleen, and thymus were fixed in 10% neutral formalin at room temperature for 24 h. After fixation, samples were processed, embedded in paraffin wax, and cut into 5-µm sections. The sections were dewaxed, stained with hematoxylin and eosin (HE), and observed by visible light microscopy. Paraffin block sections of heart, liver, kidney, spleen, and thymus were processed according to standard immunohistochemical (IHC) protocols with minor modifications. Briefly, sections were dewaxed in xylene and rehydrated with increasing concentrations of ethanol. Antigens were retrieved by immersing sections in 0.05% tween-20 and 0.01 mol/L pH 6.0 citric acid buffer for 20 min. Non-specific binding was blocked by incubation in 5% normal rabbit serum in PBS for 10 min. Slides were then incubated with rabbit anti-FAdV-4 hyperimmune serum (1:40 dilution in 0.01 M PBS, laboratory preservation) at 4 • C overnight, followed by washing and incubation with goat anti-chicken IgG/Bio (ACROBiosystems, Beijing, China) for 30 min at 37 • C and washed three times with phosphate buffer (0.05 M, pH 7.2). Immunocomplexes were detected using the 3,3-diaminobenzidine-enhanced liquid substrate system (TIANamp, Beijing, China), and the sections were counterstained with hematoxylin, air dried, and examined by visible light microscopy.

Statistical Analysis
All analyses were performed using an independent-sample t test contained in the software SAS 9.3, and a value of p < 0.05 was considered statistically significant. Results are expressed as means ± standard deviation.

Nucleotide Sequencing and Phylogenetic Analysis
A total of 195 clinical liver samples were collected from dead or diseased chickens (152 broilers, 43 layers) displaying HHS from broiler and layer flocks located in central China, in Hubei, Anhui, Jiangxi, Hunan, and Henan provinces ( Figure 1A). A total of 122 samples (93 broilers, 29 layers) were PCR positive for FAdVs. Seventy-three unique FAdVs were isolated and sequenced including 57 from broilers and 16 from layers ( Table 1). After assembly and verification, the sequences of the 73 FAdVs were submitted to GenBank (submission ID: 2262696). A phylogenetic analysis based on the obtained sequences was used to classify the 73 FAdVs into three serotypes. Fortyeight isolates of 89 positive samples were clustered in the FAdV-4 serotype sharing 68.07-97.18% nucleotide identity with hexon gene sequences of the FAdV-4 reference strain (GenBank accession No. HE608152). Twenty-four isolates from 32 positive samples were classified as FAdV-10, with 98.62-98.78% identity with the FAdV-10 reference strain (GenBank accession No. KT717889). The isolate, CH/JXJJ/1512, belonged to FAdV-2 and shared 99.84% nucleotide identity with the FAdV-2 reference strain (GenBank accession No. KT862805). The phylogenetic tree reconstruction is presented in Figure 1B.

Clinical Signs and Gross Pathology
Chickens inoculated with the CH/HBTF/1710 isolate by intramuscular injection showed obvious depression from 2 to 17 dpi. The first death occurred at 3 dpi, and at 5 dpi, mortality peaked. Four birds died at 3 dpi and 10 birds died at 5 dpi.
In the oral infection group, chickens began to show clinical signs at 3 dpi, and 12 birds died from 4 to 7 dpi. All the contact sentinel birds began to show clinical signs at 5 dpi, and one chicken died at 6 dpi. All of the surviving chickens from the intramuscular and oral groups as well as the contact sentinels, gradually recovered from 7 dpi, and no further clinical signs were observed after 17 dpi. The survival rates of birds infected by intramuscular injection, oral infection, and contact were 30, 40, and 90%, respectively (Figure 2A). During the experimental period, different clinical scores were observed in the intramuscular injection, oral infection, and contact groups. The clinical scores for oral infection were less than those of intramuscular injection at 2-5 dpi (p < 0.05) but higher at 7-11 dpi (p < 0.05). The surviving chickens from the two infection groups gradually recovered from 7 to 9 dpi, chickens in the control group showed no clinical signs, and none died during the experiment (Figure 2B). Necropsy examination of the birds at 5 and 7 dpi showed mild HHS, liver enlargement and friability, and pericardial effusion (Figures 3A,E,I). Hemorrhage and hydropericardium were detected in the heart (Figures 3B,F,J). Swelling of the kidneys and splenomegaly were also observed in all birds (Figures 3C,D,G,H,K,L). No significant gross lesions were present in the counterpart tissues of control chickens (Figures 3M-P).

Histology and Immunohistochemistry
Tissue samples from the liver, heart, kidney, and spleen were collected from the different groups, fixed, paraffinembedded, cut into sections, and stained with HE (Figure 4). No significant histologic lesions were noted in the noninfected control chickens (Figures 4D,H,L,P), while massive pathological damage was observed in tissues from the intramuscular injection group, the oral route group, and the contact sentinels.
The livers showed vacuolar degeneration and necrosis with basophilic inclusion in hepatic cells as indicated by solid arrows (Figures 4A-C). Disintegration and necrosis of myocardial fibers were observed in the heart tissue of infected chickens (Figures 4E-G). Extensive congestion in the renal interstitium and swelling and degeneration of renal tubular epithelial cells were observed in the kidneys (Figure 4K). Severe reduction and necrosis of lymphocytes was seen in the spleen (Figure 4O). Positive signals for FAdV antigen were extensively detected by immunohistochemical staining in the liver, spleen, heart, kidney, and thymus in the two infection groups and the contact sentinels at 5-7 dpi (Figures 5A-O). No FAdV antigen was detected in the control group (Figures 5P-T).

DISCUSSION
In recent years, an increasing number of IHB, HHS, and GE cases have been reported worldwide, and multiple FAdV strains have been isolated from avian species including ducks, chickens, and many wild as well as domestic birds (7,8,14,(17)(18)(19). IBH cases were first reported in 1986, in mainland China (20), but there were no reports of severe HHS prior to 2014 in China (16). However, since 2015, outbreaks of HHS have occurred with high mortality rates in chickens in most areas in China, resulting in enormous economic losses (11). In this study, 73 FAdV isolates in HHS-positive chickens from different regions in central China (Figure 1) were analyzed to determine phylogenetic relationships and virulence. Among the 73 FAdV isolates, 48 were identified and classified as serotype 4 by sequence alignment and phylogenetic analysis, 24 isolates were serotyped as FAdV-10, while CH/JXJJ/1512 was identified as FAdV-2. Interestingly, no sample in this study showed co-infection with FAdV-4, FAdV-10, and FAdV-2. Generally, FAdVs associated with IBH outbreaks were attributed to FAdV-8b, FAdV-7, and FAdV-11, while HHS cases were related to FAdV-4, based on hexon genesequence analysis. Among the FAdVs studied, 65.75% (48/73) were genetically related to serotype 4, further confirming that this serotype was the dominant agent of HHS in China from 2015 to 2018 (11,13,(21)(22)(23). FAdV-10 strains were first detected in the Shandong province of China in 2017 (24). It is worth noting that 24 isolates detected in HHS chickens in our study were serotyped as FAdV-10, with 98.62-98.78% identity to strain C-2B (serotype FAdV-10). These results indicate that poultry farmers in China may be confronted with a risk of two serotypes epidemics in the future. Fortunately, FAdV-4 and FAdV-10 were classified as the same species of FAdV-C, and the fiber-1 proteins of the two serotypes, which directly bind to the viral receptor, are highly homologous. This makes it possible to develop a diagnostic tool for detection of FAdv-4/10 (25).
The phylogenetic tree of 48 hexon sequences from outbreakassociated FAdV-4 strains obtained in this study and 13 similar strains from GenBank was reconstructed using Network 5.2 software (Figure 6). The results showed that Chinese FAdV- The pathogenicity of the CH/HBTF/1710 isolate was determined by observing the clinical signs, and the gross and histological lesions. The results demonstrated that the strain causing high mortality among 33-day-old SPF layer chickens and all birds that succumbed to infection showed serious HHS. Chickens infected by the intramuscular route showed high clinical scores and 70% mortality, which was consistent with the findings of previous studies (11,13,16). With respect to oral inoculation, the mortality approached 60%, which was higher than that obtained in previous studies (11,26). The results indicated that the CH/HBTF/1710 isolate was possibly more virulent than other strains of serotype 4, even though the outcome of experimentally induced HHS may be related to genetic differences among strains within the same serotype, the route of infection, or differential susceptibility of the chickens (6). The contact chickens presented typical HHS symptoms, and death occurred among those housed with the orally infected group suggesting that HHS is contagious and can be transmitted horizontally among chickens (5). Histopathological changes were observed in the heart, kidney, and spleen, but particularly in the liver, which revealed the presence of intranuclear inclusion bodies and multifocal necrosis. Viral antigens were detected in almost all tissues sampled from necropsied animals.

CONCLUSIONS
The results of this study showed that the FAdV-4 and FAdV-10 serotypes were involved in the outbreaks of HHS in central China from 2015 to 2018, and that the FAdV-4 serotype was the predominant one. The FAdV-4 isolates were clustered with JSJ13 and SDSX and shared an immediate common ancestor of an Indian strain. Infection of SPF chickens with the isolate CH/HBTF/1710 (FAdV-C, FAdV-4) caused HHS and resulted in a high mortality of 70%. Collectively, this study clarified the origin of FAdV-4 strains and demonstrated the genetic epidemiology of FAdV circulating in China from 2015 to 2018. These data should help researchers to develop vaccines against multiple adenovirus serotypes to control and prevent the HHS disease among domestic chickens.

DATA AVAILABILITY STATEMENT
The datasets generated for this study can be found in NCBI GenBank, NCBI Accession No. MT121992-MT122064.

ETHICS STATEMENT
Animal procedures and usage in this study were evaluated and approved by the Institutional Animal Experiment Committee from the Veterinary Faculty of Huazhong Agricultural University, Wuhan, China.

AUTHOR CONTRIBUTIONS
ZZ and DB designed research. JC and YX performed research. QX, JW, YX, and ZL contributed analytic tools. JC and ZZ analyzed data. JC wrote the paper.


s2fieldsofstudy:
Agricultural And Food Sciences
Biology

text:
A Differentially Expressed Gene from a High Oil Producer Cultivar of Castor Bean (Ricinus communis) Is Involved in the Biosynthesis of Ricinoleic Acid

Ricinus
communis or castor bean is a non-edible oilseed plant widely
cultivated worldwide for the high content of castor oil in its seeds and the
different uses the oil has in the industry. An increase in its oil content and
production efficiency is difficult, making understanding the molecular
mechanisms underlying the synthesis of oils in the seed necessary. Here, a
combined analysis of protein-protein interaction networks was performed using public
data on differential gene expression in castor bean seeds at different stages
of development. From this analysis, four key enzymes were selected and analyzed
in the polyunsaturated fatty acids pathways, whose gene expression was
subsequently quantified during the development of the seeds in a Colombian
cultivar that produces high amounts of oils and contrasted with a lower
producing cultivar. The gene coding FAH12 was differentially expressed in the
early stages of seed development in the high oil-producing cultivar and has
differences in amino acids A242V and Q319H. The analysis presents this gene as
one of those responsible for early ricinoleic acid synthesis, making it a
candidate for use in crop genetic improvement programs to increase the oil content
in castor bean.

Germplasm collections are one of the most important sources of genetic resources in the world, using which it is possible to study genetic profiles that explain the phenotypic plasticity of plants [9]. In recent years, the Colombian Agricultural Research Corporation (in Spanish, Corporación Colombiana de Investigación Agropecuaria or AGROSAVIA) has advanced in the evaluation of wild-type castor bean materials with high oil content and adaptation to tropical climates, creating a germplasm bank suitable for studying genes related to the biosynthesis of fatty acids in tropical conditions [10].
The biosynthesis of ricinoleic acid in castor bean starts with the biosynthetic pathway of triacylglycerol (TAG) in the plastids and endoplasmic reticulum (ER), a common pathway in most flowering plants that have been studied [11].
In a posterior pathway, the enzyme oleate D12-hydroxylase (FAH12, EC 1.14.13.26), located in the membrane of ER [12], catalyzes the direct hydroxylation of oleic acid (C18:1) to ricinoleic acid (C18:1-OH) [13]. The gene coding this castor bean FAH12 enzyme was already isolated and characterized [14], and Arabidopsis plants were subsequently genetically transformed, producing low levels of ricinoleic acid [15]. Thus, several studies conducted on model plants with genes related to fatty acid production pathways in seeds, have been obtained new fatty acids and unusual fatty acids, information considered the base to new studies in non-model plants [16]. This suggests in addition to key genes, other genes are also needed for the large-scale production of ricinoleic acid in seeds, such as transcription factors or accessory enzymes [17]. Evidence of the above was found when researchers studied the biosynthesis of diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) isoenzymes in castor bean in vitro plants and they identified that diricinolein and C18:1-OH-CoA are needed as substrates [18]. When these enzymes were transformed into Arabidopsis expressing the oleate D12-hydroxylase, the content of C18:1-OH in seeds increased by approximately 30% [19].
Therefore, it is necessary to increase knowledge about gene expression associated with the biosynthetic routes of fatty acids and their regulation. It could provide a better understanding of the mechanisms that control the oil synthesis and then move toward increasing their production efficiency [20]. Nowadays, high-throughput sequencing technologies make it possible to study the gene expression of the complete transcriptomes in plants [21]. This strategy was used in castor bean plants to elucidate its genome [22] and organellar genome [23] and transcriptome in tissues during seed development [24] [25]. All this genetic information helps build new knowledge-based in silico reconstruction of the interaction networks between genes, proteins, and metabolites using organism models as reference [26].
In this research, a protein-protein interaction (PPI) network was reconstructed for castor bean plant based on the orthologous proteins of Arabidopsis. Gene expression values in protein-coding genes during different seed stages were uploaded to the network and biosynthetic key genes involved in the production of fatty acids were identified and verified by RT-qPCR.
For reads mapping to the reference genome (R. communis, http://castorbean.jcvi.org/index.php) was used HISAT [28]. Annotation of the coding genes was made by comparison into the TIGR database [29]. After mapping of reads in the gene models of R. communis genome, were obtained seven files with the read counts per gene using Cufflinks version 0.9.3 [30]. Finally, differential expression ratio for each gene was calculated contrasting different stages (II to V) of endosperm development using Cuffdiff in [30]. Read counts from germinating seed, leaf and developing male flowers were pooled and taken as control.

In silico Construction and Analysis of PPI Network
We conducted a combined analysis of PPI networks (interactome) for R. communis, which was inferred by peer-to-peer orthology analysis with the proteins reported from version TAIR10. The reconstruction of the interaction network was completed by mapping orthologous proteins against the network reported for A. thaliana (ftp://ftp.arabidopsis.org/home/tair/Proteins/) using the software OrtoMCL [31]. The interaction network was enriched with the differential expression ratios obtained in Section 2.1, having fold changes in different developmental stages of the endosperm. Finally, nodes analysis corresponding to the metabolic pathways involved in fatty acid synthesis and accumulation was performed, displaying nodes (enzymes) that exhibited overexpression in the endosperm compared to the control.

Plant Material
For this research, we used cultivars of castor bean (R. communis) classified as experimental cultivar Ricinus Corpoica 03 (VERC03, Colombian collection) and experimental cultivar Ricinus Corpoica 12 (VERC12, a result of genetic improvement from Brazil). Considered as promassing cultivars after having been studied for more than a decade and that are reported as suitable to be grown in dry agoclimatic areas [10]. These cultivars were obtained from the germplasm collection at the La Selva AGROSAVIA Research Center (Rionegro, Colombia) through a Material Transfer Agreement 20162103285. The two cultivars were cultivated in an experimental field plot in the Bajo Cauca Antioqueño, Colombia (Latitude: 7˚56'29''N, Longitude: 75˚8'43''W), located at 150 meters above sea level, with 85% of relative humidity and 28˚ Celsius of average temperature during the day, the experimental area was uniformly fertilized according to [10] and irrigation at 10-day intervals. Plant material was collected for four different stages of seed development (Figure 1 and Figure S1

Fatty Acid Composition and Oil Content
Total saturated, monounsaturated, and polyunsaturated fats in castor bean seeds during different development stages were determined using gas chromatography (GC). Figure 1. Development stages of castor bean seeds selected from the VERC03 cultivar. S1 to S4 represent seed stages based on development time of seeds and their average size: image shows that S1 was defined as seeds with 10 days having 1.39 cm in average size; in S2 the seed having a minor size but with changes of testa color; in S3 stage the volume of seed increases dramatically until 1.43 cm and teste is mature with mosaic color brown and silver; S4 have capsules senesce and desiccate, and less seed weight and capsule size.  Synthesis Kit for RT-qPCR with dsDNAsa (Thermo Fisher Scientific) and cDNA products obtained were stored at −80˚C until the evaluation process.

Quantification by RT-qPCR
Primer design was made using the Primer 3 program following the parameters established by [32] seeking to amplify fragments of approximately 150 bp (Table   1) [33]. The specificity of the primers was verified using the Primer-BLAST pro-

FAD2 and FAH12 Cloning and Sequencing
Based on the analysis of PPI networks and validation by qPCR, four genes

Phylogenetic Analysis
Phylogenetic reconstruction of the desaturated/monooxygenase protein family was performed using the cloned sequence of the FAH12 gene from castor bean VERC03 to corroborate its identity. FAH12 and FAD2 gene sequences of R. communis, A. thaliana, Glycine max, and Jatropha curcas among other phylogenetically close plant species were retrieved from GenBank database (see the access numbers of the sequences in Figure 5).
Firstly, the FAH12 sequence of castor bean VERC03 was translated with Virtual Ribosome 1.1 [38] using the standard genetic code. It was added to the genes dataset in amino acids and then aligned using MAFFT v7.271 [39]. Secondly, nucleotide sequences were aligned with RevTrans2 [40]. Thirdly, these alignments were repartitioned using GBLOCKS 1 [34], and the suggested partitions were compared to the Pfam [41] annotations for the location of the active protein site. Fourthly, the best partitioning scheme was selected according to the evolution models using Partition Finder Protein 1.0.1 [42]. Finally, three topologies were reconstructed: 1) maximum likelihood with Garli (10 independent replicates supported with 1000 bootstrap pseudoreplicates), 2) maximum likelihood with RA × ML (10 independent replicates supported with 1000 bootstrap pseudoreplicates), and 3) Bayesian inference with MrBayes (2 MCMC analyses  with 10,000,000 generations with 4 chains each). All topologies were contrasted using nucleotides and aminoacid seeking to determine inconsistencies.

In silico PPI Network Construction and Analysis
A combined analysis of R. communis PPI networks (interactome) was conducted using an A. thaliana network, as a reference, composed by 11,706 proteins of (TAIR), with 24,417 interactions between these proteins. Overall, it was identified 11,192 proteins orthologous to R. communis, with significant homology to A. thaliana proteins with 23,777 interactions between them. From the protein network obtained for the castor bean plant, FAH12 protein (oleate 12-hydroxylase, genome ID 28035.m000362, E.C. 1.14.18.4) was overexpressed 10.77 times in advanced seed development stages compared with the initial development stages and other tissues ( Figure 2). Furthermore, it was evident that FAH12 also interacts with other proteins related to the production of fatty acids, establishing a possible role of this gene in the production of fatty acids in castor bean (Figure 2(b)). In addition to this gene, FAD2, DGAT2, and PDAT1-2, genes were identified, such as possible genes involved in two physiological processes: biosynthesis and fatty acid accumulation.

Fatty Acid Composition and Oil Content -VERC03 and VERC12
The analytes detected in the chromatogram of the samples were mainly classified The highest amounts of SL, ML, and PL (2.1226 g/100 g, 2.1342 g/100 g, and 2.6517 g/100 g, respectively) were detected and quantified in the fatty acids of the fruits at developmental stage II (S2) of the tropical VERC03 cultivar, revealing that the production and accumulation of triacylglycerols starts at an early stage during the formation of the fruit (Figure 3(a)). Likewise, a direct proportional relationship was determined between the quantities of fatty acids and the size of the fruit until developmental stage III (S3). Analyses of acid types 18:1-OH and 18:2-OH in the tropical VERC03 and VERC12 cultivars showed an increase in oleic and linoleic acids (Figure 3(b)).  in the stages S2 and S3 for only VERC03 cultivar (Figure 4). Expression of these genes in cultivar VERC12 (improved commercial cultivar from Brazil-Embrapa)

Quantification by RT-qPCR
showed to be statistically invariable during the three developmental stages with  respect to the UBI housekeeping gene and S1 stage as a reference. DGAT transcript was invariable in all stages and cultivars (Figure 4).

Gene Sequencing
To validate the identity of FAH12 and FAD2 genes, two fragments of 1166 bp and 1151 bp respectively, were obtained from amplification using cDNA from VERC03 cultivar, and then sequenced. The sequences obtained were aligned and comparative with sequences reported in the GenBank. Differences were found in the amino acids A242V and Q319H in FAH12 and F88V in FAD2 ( Figure S3).

Phylogenetic Analysis
A phylogenetic reconstruction was made for the desaturase/monooxygenase protein family using sequences cloned from FAH12 and FAD2 of the VERC03 castor bean cultivar and accessions retrieved from GenBank. Two groups were formed with a significant value of maximum likelihood, one with sequences of FAH12 genes and another derived from this, with FAD2 group in the top. We corroborated the identity of the clones sequenced and they were located in the same clade with each reference sequence for R. communis (Figure 5).

Discussion
In this research, we studied the gene expression related with the biosynthesis pathway of triacylglycerols in fruits of two castor bean tropical cultivars in castor bean began to take relevance from 1995 where [14] determined an increase in the differential expression of this gene in the seeds with respect to the leaves. In 2007, [20] studied the expression profiles of genes involved in fatty acid and triacylglycerol synthesis in R. communis, wherein they determined an increased expression of FAH during seed development; therefore, a direct relationship between the increase in the expression of this gene and the production Figure 5. Phylogenetic tree of FAH12 and FAD2sequences obtained from tropical castor bean seed VERC03 against other phylogenetically close plant species. Alignment was performed with MAFFT v7.271 and the reconstruction of the tree was performed with maximum likelihood using RAxML software (10 independent replicates supported with 1000 bootstrap pseudoreplicates).
of ricinoleic acid was further determined [20].  [19]. With regard to VERC12 cultivar, it was detected a progressive accumulation of polyunsaturated fatty acids during fruit maturation, which translates into a greater amount of monounsaturated and polyunsaturated fats in the last stage of development evaluated. On the other hand, seed lipids were characterized on natural castor bean mutant deficient in ricinoleic acid synthesis (OLE-1) and it was, identified high levels of palmitic and linoleic acid in the initial development seed stages, but during seed maturation, the ricinoleic acid content came up to 80% of the total fatty acids [44].
The results obtained here for DGAT2 in the VERC03 cultivar are comparable with those reported in 2010 by [27], who revealed the highest expression of this gene in the fruits at intermediate stages of development. In the same way, analyses of the expression of cDNAs in FAH12 transgenic plants revealed that the castor bean type-2 acyl-coenzyme A: diacylglycerol acyltransferase (RcDGAT2) could enhance HFAs from 17% to nearly 30%. These results indicate the probable importance of members of the DGAT2 gene family plays a key role in the process to increase the hydroxy fatty acids in castor bean [19]. Moreover, our sequence analysis of FAH12 gene led to identify two mutations, one of them (H319Q) directly related to changes in its secondary structure and also to enhance ricinoleic acid production as well as was reported by [44], who identified H319Q residue located at FAH12 His box III (domain IX), recognized as the enzyme catalytic site.
Finally, the phylogenetic reconstruction of FAD2 and FAH12 was consistent with what was reported by [14]. Our ML tree clustered both gene sequences with the same genes from Populus trichocarpa, which was also to be expected because of the taxonomic closeness of the two species. Their results suggest that FAD2 and FAH12, despite belonging to the same protein family, have a different function based on similar reaction mechanisms, which may imply an enzymatic evolution.

Conclusion
We successfully used interactome analysis, gas chromatography, qRT-PCR and gene sequencing to develop efficient methods for predicting orthology genes, analytes determination, differential expression of genes and cloning key genes involved in initial and advanced seed development stages, regarding to clarify the biosynthesis pathways and accumulation of fatty acids for a couple of tropical cultivars of castor bean. Despite the fact that we found four important genes involved in the pathway of ricinoleic synthesis in castor bean, it was possible to determine that FAH12 gene was expressed more than 3 folds in advanced seed development stages compared with the initial stages in the high oil-producing VERC03 cultivar. Additionally, it was found special differences in some aminoa-cid residues for FAH12 protein, differences involved in structural changes and enhanced ricinoleic acid production. The results obtained here are of vital importance for future genetic characterization of castor bean promising materials, thinking in using those genes and its identified mutations as molecular markers in markers-assisted breeding in castor bean plants. Finally, we suggest FAH12 gene as a key gene for plant breeding programs that seek to enhance ricinoleic acid production. Figure S1. Development stages of the castor bean seeds selected from the cultivar VERC12. S1 to S4 represent seed stages based on development time of seeds and their average size: image shows that S1 was defined by 10 days of fruit development with a white testa color; in S2 the seed have a minor size but have changes of testa color; in S3 was defined by 40 days of fruit development with a mosaic brown and black testa color; and S4 was defined by 60 days of fruit development with capsules senesce and desiccate, and less seed weight and capsule size. Figure S2. Chromatograms obtained from different development stages of the castor bean seeds selected from the cultivar VERC03.