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INFLUENCE OF PRANIC AGRICULTURE ON MORPHOLOGICAL TRAITS, CHLOROPHYLL CONTENT AND GENETIC POLYMORPHISM OF RIDGE GOURD (LUFFA ACUTANGULA L. ROXB.) ASSESSED BY RAPD MARKER ANALYSIS

Pranic agriculture is a newly emerging concept of sustainable and eco-friendly agriculture. Pranic agriculture techniques are applied on plants before sowing and at the time of plant development to improve growth and yield. The present study aimed to understand the influence of pranic agriculture on growth, yield and genetic polymorphism of ridge gourd. An increase in root length by 38%, stem girth by 8% and the number of days taken for the first harvest was advanced and found to be significant (p<.05) in pranic treated plots against the control. Chlorophyll content was 26% higher (p<.05) in pranic treated plants when compared to control. To find out the probable effects of pranic agriculture at molecular levels, RAPD marker analysis was carried out and average polymorphism upto 47% was observed between pranic and control treatments. Thus, pranic treatment was found to be very effective in increasing the overall growth and yield of ridge gourd. Further, in-depth studies are warranted about molecular mechanisms which are bringing changes in the plants after pranic treatment.

INTRODUCTION
Agriculture is a major production sector and plays a crucial role in ensuring food and nutritional security in India. In the present global climate change scenario, major challenges in agriculture production are the development of sustainable natural cropping systems by applying low-cost inputs like biofertilizers and biopesticides to reduce the burden on soil and soil microflora and to retain healthy natural ecosystem and biodiversity (Yadav et al., 2013). Among many attempts towards attaining a sustainable natural farming systems, pranic agriculture can be a complementary and supplementary method/science to the existing advanced crop production practices. Pranic agriculture is the application of prana to the plant through pranic healing techniques.
According to Master Choa Kok Sui, pranic healing is an ancient science and art of healing the bioplasmic body by using prana or life force (Sui, 2000). Prana or life force is a bioplasmic body or "Aura" which surrounds all the living organisms on earth like man, animal, plants, and microorganisms. The word bioplasmic refers to bio means life and plasma which is the fourth state of matter. Scientists with the help of Kirlian's photography have rediscovered the bioplasmic body that keeps the living organism healthy and alive (Kirlian, 1949). Plants absorb prana from sun, air, ground, and water (Sui, 2000). With limited agricultural land and an increasing human population, it is essential to enhance crop yield by improving photosynthetic activities. Photosynthesis is the foundation of life on earth providing the food, oxygen, and energy that sustains the biosphere and human civilization (Evans, 2013;Reynolds et al., 2011). Like any other animal, plants also experience abiotic and biotic stresses during its life cycle from seed germination to crop harvest. The effects of some stresses are self-repairable and some lead to heavy yield losses (Suzuki, Rivero, Shulaev, Blumwald, & Mittler, 2014;Wang, Vinocur, & Altman, 2003). Application of pranic healing on the soil before sowing, to the seed at the time of sowing and further treatment during different growth stages of the crop might lead to improved growth and yield and reverting the stress effects. Farmers can be trained to apply pranic treatment in their field and can be benefitted as it is a no-cost and no-loss method regarding the cost of cultivation is concerned.
Effect of pranic Agriculture is studied recently in some agricultural and horticultural crops like tomato, cucumber, pole beans, brinjal, papaya and found an enhancement in seed germination and vigour, plant growth, leaf area, flower number, fruit growth, yield and nutritional quality of the crop. Drumstick (Moringa olifera) seeds were exposed to Pranic treatment and grown under greenhouse conditions and seedling growth was evaluated from 15-day-old seedlings. Pranic treatment improved germination percentage and seedling vigour index as compared to control . Similarly, in Papaya (Carica papaya) pranic treatment has improved germination percentage, plant length, shoot length, number of leaves, leaf length, leaf diameter and seedling vigour index as compared to control (Prasad & Jois, 2020). In another study with Pole beans (Phaseolus vulgaris) treated with pranic agriculture showed a 3.6% higher germination percentage when compared to control. The time required for flowering and flower to fruit set was reduced by 2.2% and 3.2 % respectively in the pranic group when compared to control group (Yathindra, Jois, & D'Souza, 2017a). European cucumber pranic treated flowers started flowering earlier (5-6 days). The higher number of fruits per plant (14%) and plant yield (18%) was noticed in the pranic group when compared to control (Yathindra, Jois, Prasad, & D'Souza, 2017b). Like other vegetable crops in Tomato (Solanum lycopersicum) also there was an increase in plant height (18.5%), stem diameter (12%) and flowers per plant (31.7%) in pranic treatment as compared to control (Jois et al., 2015). Besides improvement in growth and yield, there was an increase in postharvest characteristics/qualities like low fruit water loss, shrinkage, titrable acidity and total soluble solids contents during storage both at room and cold storage conditions in pranic treatment as compared to control (Jois et al., 2015). Brinjal fruit was stored at room temperature for 0, 6, 12 and 16 days and at the end of 16 th day, pranic treated brinjal had significant improvement in firmness, non-decay, colour, gloss and non-shrivelling against control (Jois, Prasad, & Shalini, 2019). Antioxidant and polyphenol contents were found higher in pranic treated cucumber compared to control (Keerthika, Devaki, Suma, & Urooj, 2016).
Ridge gourd (Luffa acutangula) is a fruit eaten as vegetable popularly known as Kalitori, angled gourd and angled loofah, belongs to genus Luffa of Cucurbitaceae family. Ridge gourd is a famous vegetable consumed in Asian, African and Middle East countries. It is a common vegetable in the Indian diet. It is a nutritive vegetable and has a bitter taste if taken raw. Ridge gourd has been also used extensively in the Indian traditional system of medicines. Ridge gourd is reported to contain many phytochemicals such as flavonoids, saponins, luffangulin, sapogenin, oleanolic acid and cucurbitacin B (Rahman, Anisuzzaman, Ahmed, Rafiul Islam, & Naderuzzaman, 2008). Ridge gourd acts as an appetiser and it is healthy food and contains a good amount of fibre, vitamins and kind of minerals. Hence, with these above advantages, ridge gourd was chosen as one of the best Cucurbitaceous crops in the present study to find out the impact of pranic treatment on growth, yield, and chlorophyll content. More interesting and first of its kind, an attempt was initiated to understand the influence of pranic healing at the DNA level by carrying out RAPD marker analysis.

Experimental site
Ridge gourd (Luffa acutangular variety Naga) field experiment was conducted at the College of Horticulture, Mysore during February, 2019. Each treatment was carried out in a 0.2 ha land area on red loamy soil. Timely required agronomic cultural practices like weeding, irrigation, and inter cultivations were carried out according to the package of practice recommendations. The general view of the experimental plot at the time of fruiting is presented in Fig. 1.

Treatment
Pranic energy was applied to the seeds for three weeks, land area for two weeks before sowing, and to the crop two weeks after germination. Each time the pranic energy was applied for a duration of 15 minutes at twice a week interval. Another group with conventional treatment was considered as control.

Field Observations
To understand the influence of pranic treatment given to the seed, initial observations like percent germination and premature seedling death was recorded 20 days after sowing. Growth parameters like leaf area, plant height, stem girth, number of branches, root length and chlorophyll content were recorded at the vegetative stage, whereas days to the first male flower, days to the first female flower, days to 50% flower, number of male flowers, number of female flowers, percent fruit set, number of days taken for the first harvest, fruits the method of Rezadoost, Kordrostami, & Kumleh (2016) with slight modifications. The DNA quality was observed on 0.8% agarose gel and purity was checked by the A 260 /A 280 absorbance ratio using a nanodrop. The plant genomic DNA was subjected to PCR using arbitrary universal oligonucleotide primers (Table 1) for tracing genetic polymorphism. The reaction primed in a final volume of 25 µl contained the following components; DNA template: 5 µl (10 ng), primer: 2 µl (20 pmol), dNTPs: 5 µl (0.2 mM), Taq DNA polymerase: 0.5 U, PCR buffer: 2.5 µl, MgCl 2 : 1.5 mM and H 2 O: 8.5 µl. The PCR program was executed with one cycle of denaturation at 95°C/3 minutes, followed by 35 cycles of 95°C/45 seconds, 36-40°C/1 minute, 72°C/1 minute, and a final extension of 72°C/5 minutes. Amplified PCR products along with a 10 kb DNA ladder were separated by 1.8% (w/v) agarose gel and was stained with ethidium bromide and documented using a gel doc system. The percentage of polymorphism was calculated as.
Where: A = number of polymorphic bands, B = total number of bands.

Statistical Analysis
The paired t-test was carried out for the morphological and yield data using Microsoft Excel and SPSS software, and the level of significance was expressed at p=.05.

Total Chlorophyll Content
Total chlorophyll content of leaves was estimated using Dimethyl sulphoxide (DMSO) in young leaves at 55 days after sowing as given by Shoaf & Lium (1976). Fresh leaf tissue of 100 mg was cut into small pieces and incubated for 30 minutes in 7.0 ml of DMSO at 65 0 C. At the end of the incubation period, leaf tissue was discarded and also supernatant was decanted. Volume was made until 10 ml with DMSO and the absorbance of extract was read at 652 nm using DMSO as a blank. The total chlorophyll content was calculated by using the following formula and expressed as mg/g of fresh weight.
Where: A = Absorbance at specific wavelength (652 nm), V = Final volume of the chlorophyll extract (ml) and W= Weight of leaf sample (g).

RAPD Marker Analysis
RAPD marker analysis was aimed to study the effect of pranic treatment on plant DNA characteristics using a standard methodology (Babu et al., 2014). DNA was isolated separately from the young fresh leaf of ridge gourd from control (untreated) and pranic treated groups according to

Seed Germination and Premature Seedling Death
Pranic treated ridge gourd showed a higher germination percentage (89%) as compared to the control (88%). Premature seedling death was higher in the control plot (10%) as compared to the pranic plot (8.5%) (Fig. 2). There was no significant difference effect among the treatments, but higher germination and seedling survival was observed in pranic treatment than control. Treating the seeds with pranic energy before sowing might have played a role as seed priming. Seed priming is believed to bring about some biochemical changes in the metabolism within the seed, which further favors germination and growth of seedlings (Jisha & Puthur, 2014). Spiritual practices of Sriyantra, pyramid, and MahaMrtyunjayaMantra were studied on green gram and fenugreek seed germination. Green gram seeds were kept for germination in front of paper sri yantra and two models of pyramids (plywood and plastic) and the control sample was kept under the normal white paper. The Plywood pyramid has shown maximum percent emergence and radical length. Plastic pyramid has shown a maximum percentage of fresh weight. Paper Sriyantra has shown maximum percentage change in the dry weight of germinated seeds. In another experiment, fenugreek seeds are treated with MahaMrtyunjayaMantra chanting for 108 times and without chanting is considered as control. Mantra chanting has given exponential significance in seed emergence, radical length, fresh weight, and dry weight as compared to control (Jungyun, Jeeye, & Kumar, 2016).
Remarks: * Significance at 0.05 level Fig. 2. Effect of pranic treatment on growth parameters of ridge gourd

Vine and Root Growth
Ridge gourd vine length and number of branches per plant were found numerically higher in the pranic plot (6.05 m and 4.4) as compared to the control plot (5.63 m and 4.15) respectively. However, interestingly root length and stem girth were significantly higher in the pranic plot (17.82 cm and 6.48 cm) as compared to the control plot (12.87 cm and 6 cm) (Fig. 2). The root is one of the most important parts of the plant from where the plant absorbs necessary water and minerals and supplies to the above-ground plant parts. There is always an interdependent relationship that exists between the root and shoots called a source to sink relationships. Active root supplies a sufficient amount of water, nutrients, and phytohormones to shoots, and in turn, healthy leaf synthesizes photosynthates and supplies to root for its growth and uptake (Qi, Hu, Song, & Zhang, 2019). An increase in the root growth might have influenced the growth of above-ground parts like vine, leaf, and stem in pranic treatment. Another probable reason can be attributed to the increase in the synthesis of the IAA hormone in the root and shoot tips, and it helps in cell elongation and apical dominance (Paque & Weijers, 2016). This might have further lead to the increase of root length and stem girth followed by vine length and leaf area.

Chlorophyll Content
Pranic treated leaves had a high significantly of chlorophyll content (3.14) as compared to control (2.48) (Fig. 2). Chlorophyll is a green pigment and a vital component of photosynthesis. The increase in chlorophyll might have helped for the increase in photosynthesis and ultimately higher growth and yield (Kura-hotta, Satoh, & Katoh, 1987). Similarly, in ridge gourd variability and character association study regarding yield and yield attributing characters like chlorophyll was carried out by Koppad, Chavan, Hallur, Rathod, & Shantappa (2015). Chlorophyll content in the study was high in genotype Arabhavi Local 2.47 and 2.39 mg/g fresh weight at 45 and 90 DAS, respectively. Pranic energy might have improved some energy and ion equilibrium process of photosynthesis which takes place in Photosystem I and II by transfer of light energy to chemical energy and synthesis of carbohydrates and ATP. In another recent study, the combined use of energy art and energy art treated water had a significant impact in improving the growth of lettuce and bok choy plants. The impact was found in physiological development, especially in terms of carotenoid and chlorophyll content (Lee & Wu, 2019).

Flower Parameters
Flower initiation, days to 50% flowering, and number of female flowers are the most important traits for the fruit yield except for the number of female flowers per plant, all other flowering parameters are on par with each other in pranic and control groups. Whereas, the most important trait number of female flowers bearing capacity was higher in pranic treated plants (43.9) than control plants (40.1) (Fig. 3). The male/female flower ratio was less in pranic treated plants (2.35) as compared to control plants (2.45). The ratio indicates that pranic agriculture has reduced the number of male flowers and increased female flowers. A similar variation in flower ratio was found in Cucumber (Yamasaki, Fujii, & Takahashi, 2003) and Bitter gourd (Ghani et al., 2013). An increase in female flower is an important trait determining the final fruit yield per plant by increasing the number of pistillate flowers and fruit set. An early appearance of male and female flowers on the vine indicates crop earliness, reported by Tyagi, Sharma, Siddiqui, & Khandelwal (2010) in ridge gourd.

Yield Traits
Percent fruit set was high in pranic treatment (2.46%) when compared to control (2.35%) ( Table  2). The number of days taken for the first harvest was significantly lower in pranic treatment (55.25 days) as compared to control (61.5 days). In pranic treatment first fruit harvest was advanced by almost 5-6 days indicates that an increase in the number of fruit pickings and ultimately higher yield. Pranic treatment probably has advanced fruit initiation time by altering some hormones involved in flower initiation. It also might have played a role in healing the plants by damage from external oxidative stresses and lead to early recovery. Similarly, the number of fruits per plant (7.7), fruit weight (468.6), yield per plant (3.62), and yield per acre (13.4) are higher in pranic treatment as compared to control ( Table 2). The yield of a crop is decided by many of the morphological parameters, improvement in root and shoot girth and chlorophyll content might have accounted majorly for improvement in yield. The application of agri-wave technology on tomatoes remarkably stimulated seedling growth. The fresh weight of the branch, stems, and leaves of the treated tomatoes are significantly higher (59.53%) than that of the control group. The fresh weight and yield of riped tomatoes is 30.73% and 13.89% higher than untreated tomatoes (Hou & Mooneyham, 1999). In some studies, the application of biofield energy treatment has documented an increase in growth and yield compared to untreated in lettuce and tomato (Shinde, Sances, Patil, & Spence, 2012). Tyagi, Sharma, Siddiqui, & Khandelwal (2010) also reported that the number of fruits per vine had a higher positive relationship to the total yield. Long roots help in better absorption of water and nutrients from the root zone leading to the vigorous growth of the vine. By applying pranic energy there is an improvement in the capacity of two important sources of plants viz. root and leaf and ultimately leads to the increase of source to sink capacity and has increased the yield. Application of pranic energy might have promoted the metabolic activities and photosynthesis in plants in-turn enhancing vegetative growth leading to higher shoot and root growth and finally effective on overall grain yield.

RAPD Marker Analysis
RAPD analysis gel picture and results between pranic treated and control plant samples using four arbitrary primers are represented in Fig. 4 and Table  3. The DNA polymorphism bands obtained between control and pranic are represented in arrows on the left side of the lane. Totally 43 scorable bands, 20 polymorphic bands and 24 monomorphic bands were obtained between pranic and control groups. Percent polymorphism obtained by arbitrary primers GE 2, GE 3, 36.4%,53.8%,and 57.1% respectively. The percent polymorphism ranged from 36.4% to 53.8%, and the highest was observed in RPL 19A primer. The size of the amplified product varied from 400bp to 3000bp. Average percent polymorphism of 47.3% was obtained between the groups indicates that there is wide variability between the two groups. The pranic treatment has imparted considerable variability not only on plant morphology and chlorophyll content but also on plant DNA. Results of RAPD analysis give basic information and confirmation that the pranic treatment is bringing remarkable change on plants at the DNA level. The results of the present study can be a baseline for further understanding of the actual mechanism and site of action of pranic treatment on the plant at the cellular level. The additional DNA bands which are found in the pranic treated plant might be associated with the increase in the root length and stem girth of the plant. Similarly, days are taken for fruit maturity and an increase in chlorophyll content might also have some relationship with the additional polymorphic bands in pranic treated plants.  Pranic energy applied might have changed the expression of genes and protein synthesis related to the morphological and yield variations. External and internal factors bring changes in plant gene expression and regulation. External factors bring about upregulation or downregulation of the genes or receptors responsible for specific functions (Hasegawa, Bressan, Zhu, & Bohnert, 2000;Mizoguchi, Ichimura, Yoshida, & Shinozaki, 2000). In some studies, external factors like light and temperature influence gene expression related to change in photosynthetic pigment composition (Esteban et al., 2015) and flavonol accumulation (Neugart, Krumbein, & Zrenner, 2016) in different plants. All living cells can receive and process signals that originate outside their cell membranes. Similarly, pranic energy applied externally near the plant micro space might have acted as a stimulus to plants and have brought variation in gene expression related to morphology and yield. Genetic diversity studies were carried out using RAPD markers in different Cucurbitaceae family vegetables like Ridge gourd (Hoque & Rabbani, 2009), Pointed gourd (Khan, Rabbani, Islam, Rashid, & Alam, 2009), Indian ash gourd (Benincasahispida) (Pandey et al., 2008), Ash gourd [Benincasahispida (Thunb.) Cogn.] (Resmi & Sreelathakumary, 2011), Bitter gourd (Rathod, Narasegowda, Papanna, & Simon, 2008) showed 81.5%, 79.5%, 73%, 90% and 48.3% polymorphism, respectively. In another study, 93 accessions of Okra, comprise 50 West African genotypes and 43 Asian genotypes were assessed for genetic distinctiveness and relationships using the RAPD marker and they exhibited 89% similarity with nine and eight clusters in each group (Aladele, Ariyo, & de Lapena, 2008). Similarly, RAPD analysis was adopted to find out salt tolerance of segregating F 2 progenies crossed between domestic saltsensitive cultivar and natural salt-tolerant wild type Tomato parents (Ezin, Dasenka, Agbobatinkpo, Ahanchede, & Handa, 2018).
Wheat and pea seeds germinated rapidly and produced longer roots within 20 minutes by projecting 'Qi Energy'. RAPD analysis was carried out using eleven selected primers in wheat, and pea, seven primers amplified polymorphism in wheat, and five primers in pea seeds. The supply of more Qi energy during rapid cell division, growth and differentiation influenced on accelerated germination. Qi Energy might have changed the DNA at the promoter or regulatory region and brought out the changes in gene expression (Bai et al., 2000).

CONCLUSION
Pranic treated ridge gourd showed better and higher on growth, early maturity, chlorophyll content, and yield when compared to control plants. Polymorphism observed in pranic treatment over control is evident that pranic energy application bringing changes at a molecular level also. Further studies are needed to find out the underlying biochemical and molecular mechanisms behind this variation. This technique could be very promising for farmers to achieve global agriculture sustainability.

ACKNOWLEDGEMENT
Master Choa Kok Sui, the founder of Modern Pranic Healing for imparting the precise knowledge of Pranic Healing and Pranic Agriculture. World Pranic Healing Foundation, India for funding the study, Dr. Muthuraju, G.P. and Mr. Sudharshan, G.K. of College of Horticulture, Yelwala, Mysore for their technical support to carry out this work, and Mr. Papanna N. S. for application of Pranic healing techniques on plants and all those who contributed to the present study directly and indirectly.


extfieldsofstudy: Biology Medicine

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

text:
Coxiella burnetii infection in humans: to what extent do cattle in infected areas free from small ruminants play a role?

Domestic ruminants (cattle, goats and sheep) are considered to be the main reservoirs for human Coxiella burnetii infection. However, there is still a need to assess the specific contribution of cattle. Indeed, most seroprevalence studies in humans were carried out in areas comprising both cattle and small ruminants, the latter being systematically implicated in human Q fever outbreaks. Therefore, we conducted a cross-sectional study in areas where C. burnetii infection in cattle was endemic, where the density of cattle and small ruminant farms were respectively high and very low. The aim was to estimate the seroprevalence rates among two occupational (cattle farmers and livestock veterinarians), and one non-occupational (general adult population) risk groups. Sera were collected in 176 cattle farmers, 45 veterinarians and 347 blood donors, and tested for phase I and II antibodies using immunofluorescence assay. Seroprevalence rates were 56.3% among cattle farmers, 88.9% among veterinarians and 12.7% among blood donors. This suggests that a specific risk for acquiring C. burnetii infection from cattle in endemically infected areas exists, mainly for occupational risk groups, but also for the general population. Further research is needed to identify risk factors for C. burnetii infection in humans in such areas.

Introduction
Coxiella burnetii is a Gram-negative bacterium responsible for Q fever, a worldwide distributed zoonotic disease. The main animal reservoirs for human infections are domestic ruminants (cattle, sheep and goats). Q fever is as an occupational disease for people working in direct contact with potentially infected animals (e.g. livestock farmers, veterinarians and abattoir workers). However, the airborne dispersion of the bacteria also gives a potential for widespread contamination of the general population. C. burnetii is most commonly transmitted to humans through inhalation of contaminated aerosols issued from abortion and parturition products, faeces and urine of infected animals and their environment [1].
The large 2007-2010 epidemic in the Netherlands was proved to be linked to the exposure of humans to contaminated particles issued from infected dairy goat farms, experiencing abortion waves, and located in the surroundings; a large majority of cases involved the general population, while only 5% of Q fever patients had reported an activity related to agriculture [2]. In France also, the Q fever cases reported in the 2000s occurred mainly in regions with a high density of small ruminants and all the epidemiological investigations in farms in case of human outbreaks concluded about the implication of infected small ruminants [3].
By contrast, no associations between proximity or contact with infected cattle and Q fever cases in humans have been described during the period 1982-2010 in Bulgaria, France, Germany and the Netherlands [4]. Nevertheless, C. burnetii infection was reported to be widespread among Dutch dairy cattle herds (78.6%; [5]), as well as among humans living or working on dairy cattle farms (72.1%; [6]). However, the incidence rate of clinical cases among cattle farmers remained very low (0.5%) [6]. More generally, even in areas or countries where C. burnetii infection has been reported to be endemic in cattle and in humans who were occupationally exposed to cattle, the clinical cases remained rare [7]. A reason for the high level of seroprevalence in cattle farmers reported during the Dutch epidemic was that some farmers had been likely affected by the infected small-ruminant farms located nearby [6]. Thus, under the assumption that the seroprevalence rate among cattle farmers indirectly measures the implication of cattle in the spillover of C. burnetii to humans, the specific contribution of these animals might have been highly overestimated at least in the Dutch context. Indeed, a fraction of the farmers had a serological status, when positive, resulting from goat-related determinants, and being not attributable to their cattle management practices. The hypothesis of an overestimated role of cattle was supported by [8] who reported a much lower seroprevalence rate (3%) among 163 cattle farmers in Denmark, where only very few sheep and goat farms exist. To thoroughly assess the contribution of cattle, it is crucial to control this misclassification bias in the outcome of interest, due to the concomitant presence of cattle and small-ruminant farms within a same geographical area.
Therefore, we carried out a cross-sectional study purposively in areas where C. burnetii infection in cattle was endemic (to ensure putative spillover to humans nearby), where the density of cattle and small ruminant farms were respectively high and very low (to specifically assess the role of cattle), and where the annual incidence of notified clinical cases of Q fever was also very low (to assess the risk in the absence of previous outbreak), with the aim to estimate the seroprevalence rate of antibody-carriers against C. burnetii among occupational (cattle farmers and livestock veterinarians), and non-occupational (general adult population) at-risk groups. The two occupational populations were chosen in order to assess a possible exposure dose-response effect, by assuming that the durations and types of contacts with cattle differed between these two groups, the farmers being mainly exposed to one main source of infection (i.e. their herd), while the livestock veterinarians were per se exposed to multiple sources of infection and at-risk situations (such as abortions).

Study areas
The cross-sectional study was conducted from November 2017 to June 2018 in two departments (Finistère and Loire-Atlantique) located in western France (Fig. 1). These two departments met the following requested criteria to achieve the objectives: (1) a high density of cattle herds and low density of small ruminant herds; (2) no previous report of Q fever outbreaks and a low number of reported cases of human Q fever and (3) C. burnetii infection being highly endemic in cattle herds. In both departments, the density of cattle and of small ruminants were respectively high and low (>50 cows/km 2 and <3 goats and ewes/km 2 ). C. burnetii infection was endemic in cattle: the prevalence of dairy herds with seropositive animals was 69.4% in Finistère [9] and 56.1% in Loire-Atlantique respectively (unpublished data). In addition, the annual incidence of acute Q fever cases in humans was less than 1.5 cases/1 million inhabitants/year in both departments (overall mean in France: 4.5; maximum: 19) [3].

Study populations and samples
The sample issued from the general adult population consisted of blood donors from Finistère. Participants were recruited during blood donations sessions organised by the EFS (Etablissement Français du Sang). The EFS regularly organises blood collection in fixed and mobile centres all over the Finistère department that allowed the recruitment of healthy subjects with urban and/or rural living experience. For the present study, the selection of collection centres on a given date ('location-date') was made (i) on the basis of operational feasibility criteria, according to the schedule provided by the EFS, (ii) so that their location ensures a large geographical coverage of Finistère and (iii) according to the population density of the canton in which the collection took place. At each 'location-date', voluntary blood donors were offered the opportunity to participate according to age and gender quotas, which were previously defined from the characteristics of the entire population of Finistère. Thus, the resulting sample was proportional to the distribution of age, gender and place of residence (rural or urban) of the general adult population. Assuming an expected minimum prevalence of 5% of seropositive subjects in a total population of 30 000 donors in Finistère, with an error of 2.5%, the minimum number of subjects needed was 290.
Cattle farmers were recruited with the support of the GDS 44 (Groupement de Défense Sanitaire -Animal Health Serviceof Loire Atlantique) on a voluntary basis. All the dairy and beef cattle farms located in Loire-Atlantique, which housed at least 10 adult cows in September 2017, and were registered in the GDS44 database (n = 2224), were eligible and then invited by postal mail to participate in the study. Volunteers contacted the project team by email or by phone to confirm their participation. Assuming an expected minimum prevalence of 40% of seropositive farmers, with an error of 8%, the minimum number of subjects needed was 135.
All the livestock practitioners working in Loire-Atlantique and in Finistère (220 veterinarians identified from the national registration database) received by e-mail an invitation to participate in the study. Because of a low response rate at first contact, two reminders were made 1 month apart. Assuming an expected minimum prevalence of 50% of seropositive veterinarians, with an error of 10%, the minimum number of subjects needed was 50.
Whatever the populations, all participants had to fulfil the following inclusion criteria: acceptance to participate, being aged more than 18 years old, with no clinical symptoms suggestive of Q fever at the time of inclusion (both criteria being regulatory requirements to be blood donor) and with no prior history of Q fever vaccination (in the absence of any DIVA assays suited to differentiate vaccinated from naturally infected persons).

Blood collection, serum sample preparation and analysis
Cattle farmers and livestock veterinarians who agreed to participate in the study were contacted by email and/or by phone to schedule a visit at their working place for collection. First, each participant who agreed and fulfilled the sampling criteria signed informed consent to participate. With regards to blood donors, an additional tube of blood was obtained at the time of collection by the EFS nursing staff. For farmers and veterinarians, blood was collected specifically by a private nurse.
Collected blood samples were transported in a biosafety container to the Centre Clinique d'investigation at Brest hospital (INSERM CIC 1412, CHRU Brest, France) for those taken in Finistère, and to the Centre de Ressources Biologiques at Nantes hospital (BB-0033-00040, CHU Nantes, France), for those taken in Loire-Atlantique, to be centrifugated (2200 g, 15 min, 18°C) within 5-6 h of collection. Sera were then frozen and transported at −80°C to Eurofins Biomnis Clinical Trials Business unit (Lyon, France) for serological analysis. Serological analyses were performed using an immunofluorescence assay (IFA) (Focus Diagnostics, Cypress, CA, USA), to test serum samples for C. burnetii phase I and II IgG, following the manufacturer's instructions. The screening dilution was of 1:16. Participants with a positive phase II IgG result were classified as seropositive, to allow the detection of current or past infections, as IgG appears almost simultaneously with IgM in the case of C. burnetii infection and can persist over years [10]. Participants with all other outcomes were classified as seronegative.
For each population, the seroprevalence rate was calculated as the number of subjects classified as seropositive over the total number of subjects in this group.

Results
A total of 568 participants were included in the study: 347 blood donors, 176 cattle farmers and 45 livestock veterinarians. The three groups did not statistically differ according to age (mean (S.D.): 49 (15), 50 (10) and 45 (13) years-old among blood donors, cattle farmers and veterinarians, respectively). There were much more men in occupational groups compared to blood donors (48% among blood donors, 81 and 75% among cattle farmers and livestock veterinarians respectively).
The number of seropositive subjects in each group was as following: 44 blood donors, 99 farmers and 40 veterinarians. Among the 22 blood donors who worked in contact with ruminants, nine were seropositive. The corresponding seroprevalence rates are displayed in Figure 2. The estimated seroprevalence rates were higher among livestock veterinarians (88.9%; 95% confidence interval (CI): [79.7-98.1]) and cattle farmers (56.3%; 95% CI: [48.9-63.6]) than in the whole general adult population (12.7%; 95% CI: [9.2-16.2]), each of them being significantly (P < 0.05) different from the two others. Within the general adult population, the exclusion of workers in contact with ruminants resulted in a decreased seroprevalence rate (10.8%). Among all participants, only one seropositive veterinarian reported having had Q fever diagnosed by a physician in 2014.

Discussion
The present study confirmed the possible spillover of C. burnetii from cattle to humans. In infected-cattle areas almost free from small-ruminants, the prevalence rate of antibodies-carrying subjects being higher than 10%, whatever the population considered. In addition, human infection with C. burnetii is primarily occupational: the estimated seroprevalence rates were much higher in cattle farmers and livestock veterinarians than in the general adult population.
In addition, these results suggest an exposure dose-effect relationship for the risk of seropositivity. The higher the number of sources and at-risk situations (e.g. abortions with high C. burnetii shedding), the higher the seroprevalence. Indeed, it is reasonable to assume an increasing gradient in terms of cumulative exposure between the general population, the cattle farmers group, and finally the veterinarians group. The lower prevalence in the general adult population, once the workers in contact with ruminants were excluded, supports this hypothesis. A similar ranking of seroprevalence rates among veterinarians and among cattle farmers was already reported in Denmark [8].
Overall, comparisons with other seroprevalence studies must be cautious, because of different contexts (e.g. outbreak vs. endemic situation; presence or absence of small ruminants in the study area); tests (e.g. IFA vs. enzyme-linked immunosorbent assay), cut-off used (e.g. low value associated with increased sensitivity, decreased specificity and larger observed seroprevalence) or lack of information (e.g. no mention that the general population may include people working in contact with ruminants). The dilution retained in the present study to consider a participant as positive was set at 1:16, whereas a higher cut-off value of 1:32 was used in some recent cross-sectional seroprevalence studies, especially those carried out in the Netherlands, e.g. [6]. However, these studies were conducted in a post-epidemic context, while ours was performed in an endemic situation where

Epidemiology and Infection
the infectious pressure was assumed to be much lower. Therefore, the cut-off value of 1:16 that we used was purposively chosen to be lowleading to an increased sensitivity of the IFAto be able to detect infected persons with even very low IgG titres. The same choice was made by Chu et al. [11] to estimate seroprevalence among slaughterhouse workers in an endemic context in South Korea. Concerning the general adult population, the prevalence rate estimated here (12.7%) was in the range reported in studies carried out in Europe and North America (8-14%) [7]. Serological studies focusing on cattle farmers are scarce, and report either much lower estimates (<25%) [8,12] or higher (>80%) [6,13] than ours (56.3%). For livestock veterinarians, the prevalence rate found here (88.9%) was much higher than described in the literature to date (<65%) [8].
In addition, we cannot ensure the absence of selection bias having a possible impact on the estimates, while recruiting the participants in the three populations. Healthy blood donors are often considered to be poor representative of the general adult population for many chronic diseases. However, in the context of an airborne infection, we believe that this bias is highly reduced. In addition, these donors have been selected by quota sampling on age, gender and level of urbanity to make the sample as much as possible representative of the entire population of Finistère. Finally, as Q fever is often unknown of the public at large and mostly asymptomatic, it is very unlikely that the eligible donors might have chosen to participate because of a suspicion for themselves. For cattle farmers however, the risk of overrepresentation of those knowing at inclusion time that their dairy herd was positive towards C. burnetii infection cannot be excluded. This selection bias may have led to a possible overestimation of the seroprevalence rate in this group. The livestock veterinarians were also volunteers, but, as all belonged to a highly exposed group, we assume the absence of selection bias in relation to Q fever.
The present study evidences that C. burnetii infection is endemic in humans in western France. This finding may be surprising. Indeed, as the study was carried out purposively in an area with very few notifications of acute Q fever cases (only one veterinarian declared having had Q fever in the past in our study), lower seroprevalence rates might have been expected. Several reasons could explain this apparent discrepancy. First, Q fever cases may have been underdiagnosed by physicians due to its nonspecific and mostly limited symptoms, especially in a non-epidemic context as in Western France. Second, Q fever cases, when diagnosed, may be underreported to the French reference laboratory, as Q fever is not a notifiable disease in France, even if the French Public Health authorities encourage physicians to send patient serum to the French reference laboratory for diagnostic confirmation. To support these assumptions, Q fever is reported to be diagnosed predominantly in the southeast of France, where the French reference laboratory is located: thus the physician's awareness towards Q fever is highly dependent on the presence of a reference laboratory nearby [3]. Third, clinical illness for C. burnetii, when acquired from infected cattle, has been also reported to be a rare event or with a mild course [8]. Studies report that the symptomatology found in C. burnetii-infected subjects may depend on the virulence of the involved strain [14] and also on the host adaptation: C. burnetii isolates originating from infected cattle would induce in human a more pronounced proinflammatory cytokine response compared to isolates from infected goats and sheep [15]. Fourth, the magnitude of the infectious pressure to which the subjects are exposed could be another variation factor. Indeed, the seasonal reproduction in small ruminants may lead to the exposure to larger sizes of inocula possibly inhaled, compared to cattle. Finally, it is still debatable whether or not the presence of antibodies in occupationally exposed people with frequent boosting is of clinical significance [16]. All these make compatible the co-existence of high seroprevalence and low incidence rates of Q fever cases among humans exposed to cattle. The seroprevalence rates, estimated in this study conducted in an endemically infected area almost free from small ruminants, suggest that there is a specific risk for acquiring C. burnetii infection from cattle, mainly in occupational risk groups, but also for the general adult population. As already shown by Roest et al. in the Dutch context [17], comparison of strains could be performed in future research to definitively identify which types of animals (cattle vs. small ruminants vs. others) is responsible for C. burnetii infection in humans in such areas. Our finding strengthens the need to identify and quantify the impact of risk factors for C. burnetii infection in humans with the aim to determine specific preventive actions which could be advised to the human populations living in contact with or close to cattle.


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Simultaneous Biofortification of Rice With Zinc, Iodine, Iron and Selenium Through Foliar Treatment of a Micronutrient Cocktail in Five Countries

Widespread malnutrition of zinc (Zn), iodine (I), iron (Fe) and selenium (Se), known as hidden hunger, represents a predominant cause of several health complications in human populations where rice (Oryza sativa L.) is the major staple food. Therefore, increasing concentrations of these micronutrients in rice grain represents a sustainable solution to hidden hunger. This study aimed at enhancing concentration of Zn, I, Fe and Se in rice grains by agronomic biofortification. We evaluated effects of foliar application of Zn, I, Fe and Se on grain yield and grain concentration of these micronutrients in rice grown at 21 field sites during 2015 to 2017 in Brazil, China, India, Pakistan and Thailand. Experimental treatments were: (i) local control (LC); (ii) foliar Zn; (iii) foliar I; and (iv) foliar micronutrient cocktail (i.e., Zn + I + Fe + Se). Foliar-applied Zn, I, Fe or Se did not affect rice grain yield. However, brown rice Zn increased with foliar Zn and micronutrient cocktail treatments at all except three field sites. On average, brown rice Zn increased from 21.4 mg kg–1 to 28.1 mg kg–1 with the application of Zn alone and to 26.8 mg kg–1 with the micronutrient cocktail solution. Brown rice I showed particular enhancements and increased from 11 μg kg–1 to 204 μg kg–1 with the application of I alone and to 181 μg kg–1 with the cocktail. Grain Se also responded very positively to foliar spray of micronutrients and increased from 95 to 380 μg kg–1. By contrast, grain Fe was increased by the same cocktail spray at only two sites. There was no relationship between soil extractable concentrations of these micronutrients with their grain concentrations. The results demonstrate that irrespective of the rice cultivars used and the diverse soil conditions existing in five major rice-producing countries, the foliar application of the micronutrient cocktail solution was highly effective in increasing grain Zn, I and Se. Adoption of this agronomic practice in the target countries would contribute significantly to the daily micronutrient intake and alleviation of micronutrient malnutrition in human populations.

INTRODUCTION
Globally, an estimated 2 billion people suffer from micronutrient malnutrition (also known as hidden hunger), especially in the developing world. Particular micronutrient deficiencies include Fe, Zn, Se and I which result in severe health complications (Bailey et al., 2015;Harding et al., 2018) as well as significant economic losses (Gödecke et al., 2018). Due to their high requirements, infants, preschool children, and women of reproductive age are more vulnerable to micronutrient malnutrition.
Iron deficiency has been suggested to be the most common micronutrient disorder in human populations, and is often associated with anemia (Bailey et al., 2015). The most prevalent form of anemia is the Fe deficiency anemia which affects over 1 billion people worldwide (Camaschella, 2019). Zinc malnutrition has been found to be responsible for the death of 116,000 children under five years of age, mainly due to enhanced infectious diseases such as diarrhea and pneumonia (Black et al., 2013). Zinc has strong antiviral and antibacterial effects in the human body and is involved in improving immune function and immune response against various viruses, including corona virus (Read et al., 2019;Skalny et al., 2020). Similarly, Se is also required for optimizing the immune system against viral attacks and is required in prevention of various types of cancer in human body and can lower risk of some cancers (Steinbrenner et al., 2015). A large proportion of the world population is known to be at risk of Se malnutrition due to inadequate dietary Se intake (Jones et al., 2017).
Despite the use of iodized salt since decades, human I deficiency has been reported to be still prevalent in a number of countries. An estimated 2 billion people are at risk of I deficiency (Zimmermann and Andersson, 2012;Pearce et al., 2013). Even, some reports indicate that I deficiency is a re-emerging public health problem (Lazarus, 2015;Panth et al., 2019). The welldocumented role of I in the human body is related to biosynthesis of thyroid hormones which are important in brain function and mental development (Pearce et al., 2016).
Low availability of soil micronutrients to crop plants and consequent inadequate dietary intake of micronutrients have been discussed as the root cause for the high incidence of micronutrient deficiencies in humans (Welch et al., 2013). According to Welch et al. (2013) crop production systems have never been designed with the aim to improve human nutrition and health which is a further particular reason which partly explains the widespread occurrence of hidden hunger in the world. Therefore, crop production systems should include nutrition-sensitive approaches and be aligned with the production of micronutrient-enriched (biofortified) staple foods for the target populations (Bouis and Saltzman, 2017;Cakmak and Kutman, 2018;Jha and Warkentin, 2020). Most of the staple foods are inherently very low in micronutrients and cannot meet the recommended daily intake of micronutrients (Cakmak, 2008;Andersson et al., 2017;Bouis and Saltzman, 2017), especially rice which is inherently low in micronutrients and is then processed extensively before consumption such as polishing, steaming and parboiling (Balbinoti et al., 2018;Ito and Lacerda, 2019). Today, increasing nutritional value and composition of rice, especially regarding micronutrients is, therefore, a growing challenge.
Among the approaches for enrichment (i.e., biofortification) of micronutrient in staple foods, genetic (i.e., plant breeding) and agronomic (i.e., use of fertilizers) approaches are used widely. After long-term successful efforts, the International HarvestPlus program has developed and released new Zn-or Fe-biofortified wheat and rice varieties containing additional Zn or Fe, at about 10 mg per kg grain (Andersson et al., 2017;HarvestPlus, 2020). Also, multi-country field trials established in the framework of the HarvestZinc project have demonstrated that agronomic biofortification, a fertilizer-based approach, is highly effective in improving grain concentrations of the targeted micronutrients in food crops as shown in wheat, rice, maize and bean for Zn, Se and I (Phattarakul et al., 2012;Zou et al., 2012;Mao et al., 2014;Ram et al., 2016;Cakmak et al., 2017). The impact of the fertilizer-use strategy on grain micronutrient concentration was much more pronounced for foliar in case of the use of foliar sprays, compared with soil applications, for example, Zn in rice plants grown under field conditions (Wissuwa et al., 2007;Phattarakul et al., 2012;Saha et al., 2017). Higher agronomic effectiveness of foliar application over soil application in boosting grain Zn has been also shown for several other crop species (Wang et al., 2012;Zou et al., 2012;Mao et al., 2014;Cakmak and Kutman, 2018).
The contribution of root absorption and root-to-grain transportation of Zn and Fe during the grain filling stage is minimal under field conditions compared to the greenhouse conditions with always high amount of available micronutrients and optimal water (Waters et al., 2009;Kutman et al., 2012;Sperotto et al., 2013;Cakmak and Kutman, 2018). This indicates that mobilization and retranslocation of the already deposited micronutrient reserves within the vegetative tissues to the grains are the key constraints affecting grain accumulation of micronutrients under field conditions. Therefore, maintaining a high pool of micronutrients in the vegetative tissue just before and/or during the grain filling stage, which can be attained with foliar application of micronutrients during grain filling stage, is of great importance and relevance for increasing grain micronutrients (Cakmak and Kutman, 2018). As shown for rice, plant genotypes differ in their genetic capacity to mobilize and translocate Zn from vegetative tissues to grains which needs to be considered in micronutrient biofortification studies Johnson-Beebout et al., 2016).
In the present study, we aimed to investigate the foliar applications of Zn, I, Fe and Se alone or together in a cocktail solution on the grain concentrations of these micronutrients in rice grown under different soil and crop management conditions of five major rice growing countries over two years by using seven different rice cultivars. Indeed, there are many publications about the effect of soil and/or foliar applications of micronutrients, usually alone or 2 combined elements, on grain concentration of micronutrients in food crops (Cakmak, 2008;Duffner et al., 2014;Dimkpa and Bindraban, 2016;de Valença et al., 2017;Lyons, 2018). To the best of our knowledge, this is the first study investigating the effect of a cocktail micronutrient solution on grain concentrations of Zn, I, Fe and Se under diverse soil and environmental conditions in five rice growing countries.

Establishment of Field Experiment
A field experiment on rice was conducted over two cropping seasons in 2015 to 2017 at a total of 21 field sites of five countries, i.e., Brazil, China, India, Pakistan and Thailand. Except for Brazil, where rice was cultivated under upland conditions, in all other locations rice was grown under flooded conditions with 10 to 20 cm standing water above the soil surface for most of the growing season. The rice cultivars grown at various field sites were commonly cultivated in the respective countries ( Table 1). Initial soil characteristics of the field locations, including DTPAextractable soil Zn and Fe measured by Lindsay and Norvell (1978), I determined by Cakmak et al. (2017) and Se measured by Dhillon et al. (2005) are presented in Table 2. The basal fertilizers applied according to local practices of the countries are given in Table 3 and were accomplished before transplanting the crop (except for Brazil, were rice seeds were used instead of seedlings). The given rates of N fertilizer were split-applied and based on respective country's recommendations as detailed in Table 3.

Experimental Design, Foliar Applications and Crop Harvesting
The experiment comprised four treatments: i) local control (i.e., basal N, P, K fertilizers only, no micronutrient applied); ii) local control + foliar application of Zn (0.5% ZnSO 4 ·7H 2 O in spray solution); iii) local control + foliar application of I (0.05% KIO 3 in spray solution); and iv) local control + simultaneous foliar spray of micronutrients (Zn, I, Fe and Se) in a cocktail solution containing 0.5% ZnSO 4 ·7H 2 O + 0.05% KIO 3 + 0.02% Fe-EDTA + 0.001% Na 2 SeO 4 . The field experiment was laid out in a randomized complete block design with four replications. The foliar solution of each treatment, varying between 500 to 800 L ha −1 , was applied twice, i.e., at the panicle initiation (about one week prior to heading) and the early grain milk stages. The foliar treatments were applied at late afternoon time until the solution started to run-off from the leaves as described by Cakmak et al. (2010). At grain maturity, rice grain yield was recorded from 6 to 10 m 2 central area of each plot.

Chemical Analyses
Subsamples of brown rice (whole caryopsis, without husk) from each plot were secured for analysis of Zn, I, Fe and Se. Brown rice samples were rinsed and washed with distilled de-ionized (DDI) water and dried first with tissue papers and then at about 45 • C in a forced-draft oven to constant weight. Dried brown rice grains were digested in HNO 3 -H 2 O 2 mixture in a microwave accelerated reaction system (CEM Corp., United States) for the determination of Zn, Fe and Se. For I analysis, brown rice samples were extracted in TMAH at 90 • C using a closed-vessel microwave reaction system (CEM Corp., United States) as described earlier by Zia et al. (2015) and Cakmak et al. (2017). Zinc and Fe concentrations in the digested solutions were measured by inductively coupled plasma optical emission spectroscopy (ICP-OES), and I and Se concentrations by inductively coupled plasma mass spectrometry (ICP-MS). Measurement of mineral nutrients was checked by using certified standard reference materials obtained from the National Institute of Standards and Technology (Gaithersburg, MD, United States).

Statistical Analysis
The significance of the effects of different foliar treatments on the dependent variables was determined using one-factor ANOVA at 0.05 probability level of least significant difference (LSD) test using SAS software (SAS 8.0, United States). For overall effectiveness, the data sets across locations and years were Extractable I was determined by Tetra methyl ammonium hydroxide (TMAH) method by Zia et al. (2015) and Cakmak et al. (2017), extractable Se was determined by Dhillon et al. (2005), extractable Zn and Fe were determined by DTPA, extractable P by Bray-II and extractable K by NaHCO 3 .

Grain Yield
With the same experimental treatments, rice grain (paddy) yields varied drastically across 21 field sites across 14 locations in five countries. For example, in case of the local control treatment (i.e., no micronutrient application), rice grain yield varied from 1.94 Mg ha −1 at Lambari location of Brazil in 2017 to 11.46 Mg ha −1 at Jiangsu location of China in 2015 (Table 4). Foliar application of Zn, I or micronutrient cocktail did not affect rice grain yield at any field site in any country. Across all experimental treatments and field sites in five countries, average grain yield was 6.10 Mg ha −1 with the local control treatment, 6.26 Mg ha −1 with the foliar Zn treatment, 5.95 Mg ha −1 with the foliar I treatment, and 6.06 Mg ha −1 with the micronutrient cocktail treatment ( Table 4).

Grain Zn, I, Fe and Se Concentration
With the local control treatment, brown rice Zn concentration ranged from 15.0 mg kg −1 at the Ludhiana field site of India in 2017 to 26.9 mg kg −1 at CMU field site of Thailand in 2015 ( Table 5). Application of foliar Zn and micronutrient cocktail significantly increased Zn concentration in brown rice at all Nd, no data (due to an experimental error in the field, grain yield could not be collected). NS, non-significantly different at P < 0.05.
field sites in all countries (P < 0.05), except for at Ludhiana field site of India in both years and at Gurdaspur field site of India in 2016 (Table 5). Average increase in brown rice Zn concentration, across all field sites in five countries, was 31.3% with foliar Zn application and 25.2% with foliar micronutrient cocktail application. The range in I concentration in brown rice was wider than the range in Zn concentration. In the case of the local control treatment, I concentration varied from 4 µg kg −1 at Ludhiana and Gurdaspur field sites of India in 2016 and 2017, respectively to 34 µg kg −1 at Patos de Minas field site of Brazil in 2017. Foliar application of I and micronutrient cocktail resulted in several fold increases in brown rice I concentration at all field sites in all countries (P < 0.05; Table 6). On average, across all field sites in all countries, rice grain I concentration was 11 µg kg −1 with the local control, 204 µg kg −1 with the foliar I, and 181 µg kg −1 with the foliar micronutrient cocktail applications.
Variation in Fe concentration in brown rice across all field sites with the local control treatment was greater than the variation in grain Zn concentration. With this treatment, brown rice Fe concentration showed a range between 8.0 mg kg −1 at Gurdaspur field site of India in 2016 and 27.8 mg kg −1 at Jiangsu field site of China in 2015 (Table 7). However, unlike brown rice Zn density, rice grain Fe concentration was not affected by foliar application of micronutrient cocktail, except for two field sites in Brazil, i.e., Lambari in 2016 and Patos de Minas in 2017. Consequently, average brown rice Fe concentration with all experimental treatments remained almost the same, i.e., 11.8-12.8 mg Fe kg −1 grains ( Table 7).
The Se concentration in brown rice across all field locations showed a large variation with the same micronutrient treatment. With the local control treatment, it was 3 µg Se kg −1 at Patos de Minas, Brazil in 2016 to as high as 404 µg Se kg −1 at Ludhiana, India in 2017 ( Table 8). The foliar application of the micronutrient cocktail resulted in substantial increases in Se concentration in brown rice at all field locations in all countries (P < 0.05); the overall magnitude of increase in grain Se concentration was 4-fold (Table 8).

Relationships Between Rice Grain and Soil Concentrations of Zn, I, Fe and Se
The relationships between soil extractable micronutrient concentrations and their grain concentrations are presented in Figure 1. There was even no correlation between soil extractable concentrations of I, Fe and Se and the concentrations of these micronutrients in rice grain. In fact, there even a negative relation between soil extractable Zn and grain Zn concentration in rice (R 2 = 0.17, P < 0.05).

DISCUSSION
Results of the present study conducted across 21 field sites over two crop seasons in five major rice-growing countries showed that the foliar application of a micronutrient cocktail solution Nd, no data (due to an experimental error in the field, grain samples could not be collected). NS, non-significant different at P < 0.05. Numbers in the same row followed by different letters differ significantly at LSD 0.05 . * = significantly different at P < 0.05.
was highly effective in increasing brown rice grain concentrations of Zn, I and Se. The increases in grain concentration of micronutrients happened without any clear change in grain yield by application of the cocktail solution of micronutrients ( Table 4). The soils of all the field sites were not deficient in Zn and Fe for rice, and contained generally high extractable concentrations of Zn and especially Fe (Sims and Johnson, 1991). The DTPA-extractable soil Zn and Fe concentrations ranged between 0.6 to 3.7 mg kg −1 and 14 to 176 mg kg −1 , respectively ( Table 2). Although several soil chemical and biological factors are known to affect the interpretation of the effect of soil DTPAextractable Zn on plant growth, it can be, however, generalized that the rice plants may respond to Zn fertilization positively if the DTPA-extractable Zn is below 0.5 mg kg −1 (Sims and Johnson, 1991;Fageria, 2013;Duffner et al., 2014). Also, foliar application of Zn is less effective in increasing grain yield of crops compared with its soil application . Lack of grain yield increase in response to foliar application of Zn and Fe across these field sites could be attributed to adequate amount of DTPA-extractable Zn and Fe for rice ( Table 2).
To our knowledge, this study is the first one showing a very positive effect of combined foliar sprays of Zn, I and Se on grain concentrations of these micronutrients in many rice cultivars grown in several countries. A similar positive effect of the foliar applied micronutrient cocktail solution on grain concentrations of the micronutrients has been also shown in wheat grown in 6 different countries (Zou et al., 2019). In contrast to Zn, I and Se, the concentration of Fe in grain was not affected with the spray of the micronutrient cocktail (Table 7). There was only a minimal increasing trend in grain Fe (by about 8%) by spray of micronutrient cocktail solution. Also, foliar applications of Zn to rice foliage in both single and cocktail solutions were also effective in increasing Zn concentration of brown rice grains at most of the field sites (P < 0.05; Table 5). The positive effect of foliar application of Zn alone on grain Zn has been shown already in rice (Phattarakul et al., 2012;Mabesa et al., 2013;Goloran et al., 2019). In the present study, the positive impact of foliar Zn application on grain Zn was consistent during both years at all field sites in five different countries, with the exception of a few field sites in India (Table 5). There was, however, a distinct genotypic variation in response to foliar Zn fertilization. For example, the cultivar Zhengdao11 from the Jiangsu location in China consistently showed the highest response to foliar Zn fertilization over 2 years and exhibited an increase over 50%, while the cultivar Xiyou19 from Chongqing in China showed an increase of around 17% only ( Table 5). Both rice cultivars used in India (i.e., PR 124 and PR 121) were less responsive to foliar Zn application in terms of increasing grain Zn. It is well-documented that rice cultivars show a large variation in their ability to translocate and deposit Zn in the grains Johnson-Beebout et al., 2016;Sanjeeva Rao et al., 2020). The weak of relationship between grain Zn and DTPAextractable soil Zn (Figure 1) indicates that retranslocation of Zn from the vegetative parts to grains, rather than the root Zn uptake, might be a key process affecting grain Zn accumulation in different rice genotypes grown in five countries. In agreement with this suggestion, Mabesa et al. (2013) and Impa et al. (2013) showed that grain Zn accumulation in rice genotypes is under a significant influence of Zn remobilization and transport from vegetative parts to grains. However, the relationship between the concentrations of two mineral nutrients existing in the soil and grain depends not only on their chemical availability, but also on the available amount of other mineral nutrients in soil. It is well documented that there is antagonistic interaction among the mineral nutrients during their root uptake and root-to-shoot transport which greatly affects the leaf and grain composition of mineral nutrients (Marschner, 2012;Rietra et al., 2017). Therefore, the weak correlation between the micronutrients in soil and grain (Figure 1) may also depend on the antagonism between nutrients during root uptake. In case of some rice genotypes grown under greenhouse conditions, grain Zn has been found to be in good correlation with the DTPA-extractable soil Zn (Fageria et al., 2011;Johnson-Beebout et al., 2016). Based on these results, it has been suggested that keeping adequate plant available Zn in the soil medium until grain maturation would contribute greatly to grain Zn accumulation through continued and direct root uptake of Zn with little reliance on retranslocation of Zn from vegetative tissues (Johnson-Beebout et al., 2016;Cakmak and Kutman, 2018). Generally, grain Zn correlates very positively with the amount of Zn in the growth medium if Zn is continuously supplied or remains available to the plant roots until grain maturation, which is very common under controlled greenhouse or growth chamber studies. By contrast, when soil Zn availability to plant roots is limited especially during reproductive growth stage, which is very common under field conditions, grain Zn accumulation largely depends on remobilization of Zn from vegetative tissues (Stomph et al., 2009;Waters et al., 2009;Cakmak and Kutman, 2018). Therefore, it can be suggested that the rice genotypes with higher mobilization and translocation capacity for Zn (and also other micronutrients) from the vegetative tissues to grain are potentially the most promising genotypes for higher grain Zn accumulation.
It was important to note that across all field sites, the average Zn concentration in rice grains with foliar application of Zn alone (i.e., 28.1 mg kg −1 ; Table 5) was very similar to the increase achieved by the cocktail spray of micronutrients (i.e., 26. 8 mg kg −1 ). This result clearly indicates that when applied together with three other micronutrients, the leaf absorption and transportation of Zn in the grain are not seriously affected by the other micronutrients present in the same spray solution. Foliar application of KIO 3 alone had no effect on rice grain Zn and even  Numbers in the same row followed by different letters differ significantly at LSD 0.05 . * = significantly different at P < 0.05. Nd, no data; NS, non-significant different at P < 0.05. Numbers in the same row followed by different letters differ significantly at LSD 0.05 . * = significantly different at P < 0.05.
Frontiers in Plant Science | www.frontiersin.org tended to increase grain Zn (Table 5). A very similar finding has been also reported in field studies conducted on wheat grown in six countries (Zou et al., 2019). The lack of antagonism between Zn and Se (Mangueze et al., 2018) or Fe and Zn (Wei et al., 2012) has been also shown in rice when these micronutrients are sprayed together in the same spray solution.
Iodine is not an essential micronutrient for higher plants (Marschner, 2012) but, food crops absorb, transport and accumulate I in edible parts (Fuge and Johnson, 2015;Cakmak et al., 2017;Gonzali et al., 2017). There are a few studies indicating that I has some beneficial effects on plant growth, however, I might be also phytotoxic when applied at higher concentrations (Medrano-Macías et al., 2016;Gonzali et al., 2017). In the present study, substantial increases were found in grain I with the foliar application of KIO 3 ( Table 6). This significant effect of KIO 3 on grain I was consistent over two years across the 21 field sites (P < 0.05) and was observed with the application of I alone or in combination with other micronutrients in the cocktail. There were no clear antagonistic effects of other micronutrients on grain I accumulation when sprayed together ( Table 6). Our earlier studies showed that foliar application of KIO 3 to rice grown under field conditions in Brazil and Thailand was effective in increasing grain I concentration, and similar positive effects were also found with the foliar application of KI (Cakmak et al., 2017). These results suggested that I is transported to grains via the phloem channel. There are, however, different opinions and discussions regarding phloem mobility of I. Very recently, Golob et al. (2020) showed that foliar-applied I either in the form of KI or KIO 3 significantly enhanced I concentration in the tubers of kohlrabi plants, indicating phloem transport of I in the plants. Similar findings related to phloem transportation of I have been also reported by Smoleń et al. (2016) and Landini et al. (2011). By contrast, there are studies showing poor phloem transport of I in the plants such as in young spinach plants (Humphrey et al., 2019) and apple trees (Budke et al., 2020). In case of the studies with apple trees, the increases in I concentration of the fruits, which were kept in plastic bags during the foliar spray, were around 4-to 5-fold compared to the trees without foliar I spray (i.e., from 0.4 µg up to 2.0 µg per 100 g FW) (Budke et al., 2020). These increases were, indeed, very significant; but the dietary relevance seems to be minimal. As indicated earlier, for cereals, at least a part of the enhanced grain I through foliar spray could be also related to a direct fortification (i.e., contamination) of florets and seeds with I via spray solution (Cakmak et al., 2017). The differences among the plant species in their capacity for transporting I from vegetative tissues to the sink organs (i.e., grains, fruits, tubers) are probably related to the plant species-related factors, concentration of I in spray solution and coverage uniformity of the I spray solution on the leaves. The age of the leaves (i.e., sink or source status of the leaves) of the experimental plants used in the short-term studies might be also another factor. Considering published data and reported large gradient in I concentrations between the sink and source organs (Tsukada et al., 2008;Cakmak et al., 2017;Budke et al., 2020), it can be suggested that I is not highly phloem mobile in the plants, like nitrogen, phosphorus or potassium (Marschner, 2012); but it can be considered as moderately phloem mobile as also suggested by Hurtevent et al. (2013). Further studies are required for better clarification and understanding of the phloem mobility of I in different plant species considering source and sink status of the treated leaves and other factors highlighted above.
The results in Table 6 suggest that grain I concentrations were similar between the foliar sprays of I alone and the cocktail of the micronutrients. As mentioned earlier, there is no antagonistic interactions between Zn and I during their transport and grain accumulation when sprayed together in the same solution. Similarly, also I and Se seem to be not antagonistic ( Table 6). Also in previous studies, no clear interaction was found between I and Se during their leaf absorption and transportation to wheat grains (Zou et al., 2019), lettuce roots (Smoleń et al., 2014), kohlrabi tubers (Golob et al., 2020) and apple fruits (Budke et al., 2020).
The foliar application of the micronutrient cocktail solution was also effective in increasing grain Se concentration at all field sites in five countries (P < 0.05). On average, the increase in grain Se with foliar cocktail treatment was 4-fold (i.e., from 95 µg kg −1 to 380 µg kg −1 ; Table 8). Selenium is known to be highly phloem mobile in plants, and therefore foliar spray of Se at relatively low rate is suggested to achieve adequate levels of Se in food crops for human nutrition (Lyons, 2018). Considering the Se concentration in micronutrient cocktail of the present study (i.e., 0.001% Na 2 SeO 4 ) and two foliar sprays (@ 600 L cocktail solution ha −1 ) the amount sprayed was about 5 g Se ha −1 which resulted in marked increases in grain Se. As rice is a predominant staple cereal in many countries of the world, especially in developing countries, Se-enriched rice through the agronomic biofortification could be a sustainable Se source for the human populations living in Se-deficient regions.
In contrast to Zn, I and Se, foliar spray of the micronutrient cocktail solution did not affect grain Fe concentration compared with the local control treatment, except for one field site in Brazil, i.e., at Lambari in 2016 (P < 0.05; Table 7). Usually, there is little change in grain Fe concentration in response to foliar Fe application which was ascribed to poor mobility of Fe in phloem (Aciksoz et al., 2011;El-Jendoubi et al., 2014). In a field experiment conducted on several Fe-rich rice cultivars in China, Yuan et al. (2013) observed an average increase of about 15% in Fe concentration of brown rice with foliar-applied Feamino acid complex (Fe-AA) alone, and 33% increase when 1% (w/v) nicotianamine was added in the Fe-AA solution (P < 0.05). By applying the same micronutrient cocktail solution used in the present study, Zou et al. (2019) found that grain Fe concentration increased only by 13% in wheat grains grown in 27 locations of 6 countries over 2 years. The effect of foliar Fe sprays on grain Fe reported in the literature is very variable and, usually, of little biological significance (Rengel et al., 1999;Aciksoz et al., 2011;Niyigaba et al., 2019). Due to very low phloem mobility of Fe, transgenic approaches might be useful for enrichment of rice grains with Fe to adequate levels for human nutrition Huang et al., 2020).
White rice (polished rice) represents the common form for human consumption; however, its nutritional value is lower than brown rice due to loss of significant amount of minerals during polishing, especially micronutrients such as Fe (Promu-thai et al., 2007;Hansen et al., 2012;Balbinoti et al., 2018). Therefore, a decline in the micronutrient concentrations of rice grain is expected after the polishing process, but the extent of the loss depends on rice cultivars, distribution pattern of the mineral nutrients within grain as well as the degree of milling in polishing process (Prom-u-thai et al., 2007;Hansen et al., 2012;Saenchai et al., 2012).

CONCLUSION
Foliar application of the cocktail micronutrient solution resulted in significant enrichments of Zn, I and Se in brown rice grains under a wide range of environmental and soil conditions of five major rice producing countries. Based on the data published by Statista GmbH the studied five countries produce almost 300 million metric tons of milled rice which corresponds to about 60% of world production of milled rice per year (Shahbandeh, 2020). Thus, a successful adoption of this agronomic strategy at least in the five major rice producing countries would contribute significantly to the daily micronutrient intake and alleviation of micronutrient malnutrition. Very recent studies using a simulated human digestive system showed that Zn, I and Se in agronomically biofortified foods are sufficiently bioaccessible for use by the human body (Cakmak et al., 2020). In a further human Zn absorption study conducted at ETH-Zurich, agronomically biofortified wheat with Zn made a significant contribution to the dietary Zn absorption in humans (Signorell et al., 2019). These new results indicate an effective transfer of micronutrients from field to gut, and highlight relevance of the fertilizer use strategy in fighting the global burden of micronutrient malnutrition in human populations.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.


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Production and nutritive value of silage corn in different reproductive stages

Corn has been the most used crop for whole plant silage, due to its high potential for dry matter production, high energy and great adaptability to different growing regions. Due to the great importance of the culture for this purpose, the region of the Far West of Santa Catarina, due to the presence of one of the largest dairy basins in Brazil, demands the supply of this food to the animals mainly during the season of forage. Thus, the objective of this work was to evaluate different hybrids and to identify the optimal corn cutting time for silage production that provides a greater economic return to the system. The experimental design used was a randomized complete block design in a 2x4 factorial scheme, allocating different maize hybrids (P30F53 VYHR®; P32R48 VYHR®) to the main plot and the reproductive stages (R2, R3, R4, R5). The experiment had 8 treatments and 3 replicates, totaling 24 experimental units. The studied variables were: dry matter content (DM); crude protein (PB); acid detergent insoluble fiber (FDA); neutral detergent insoluble fiber (NDF); total digestible nutrients (NDT), and CNF-amido, at the reproductive stages. The best economic yield is obtained when the maize crop is ensiled at the reproductive stage R4 and R5. Among the hybrids, the P32R48 VYHR® was the one that obtained better yield, providing greater production of milk by

I. INTRODUCTION
The importance of dairy monoculture in the state of Santa Catarina is indisputable, both as a source of income for rural properties, as for the generations of jobs in the processing industries. Currently, the state is the fourth largest milk producer in the country, with 2.83 billion liters of milk, with emphasis on the western region of Catarinense, which represents 79% of this production (EPAGRI/CEPA, 2018). To maintain this growth, producers constantly seek, forms of supply the nutritional demands of their herds, with quality pastures, supply of concentrated feedingstuffs or preserved foods (silage or hay). In this sense, it gains prominent role, the making of corn silage plant. Due to food shortages during certain periods of the year, when the forage used as the basis for feeding livestock, cannot meet the nutritional demands. Maize has been the most used crop for making plant silage whole due to its high potential for dry matter production and its large adaptability to different cultivation regions (FACTORI et al., 2012). Still, there is a large number of maize hybrids destined for silage production, from which significant differences with respect to the production of Green forester, production of grains, leaves and stalk directly interfering with the nutritional value of the silage (MORAES et al., 2013). In this sense, the choice of hybrid and reproductive stage of cutting becomes determinant factors for the success of the system, since the hybrid may represent up to 50% of the final yield (FACTORI et al., 2012), and the stage of cutting that the plant is in, is directly related to the available energy of this material, by increasing the participation of grains (MARAFON et al., 2015). In this context, it is important studies, which demonstrate the best strategy for harvesting these hybrids, which provide higher milk production ha-1 and economic income for the rural producer. In this way, the objective of this work was to evaluate different hybrids and identify the ideal moment of cutting of maize for silage production that provides greater economic return to the system.. The base fertilization used was 520 kg ha-1 of the fertilizer MAP (11-52-00), the pre-planting fertilization (21 days before sowing) was 800 kg per hectare of potassium chloride 00-00-60, and in the fertilization of coverage were used 320 kg per hectare of the fertilizer nitrogenated urea (45-00-00), at V4 Stage, and 480 kg per hectare of Sulfammo MeTA® (29-00-00), in the V6 stadium.

II. MATERIALS AND METHODS
The hybrids used were the Pioneer brand, P30F53 VYHR® and P32R48 VYHR®, both with Leptra® technology (maize cartridge caterpillar, Spodoptera Frugiperda) and RR (Roundup Ready®), with industrial seed treatment (Dermacor® + Poncho® + Derosal Plus®). Weed control was performed 20 days after sowing of the crop with a herbicide based on Atrazine + Simazine (Primatop® 6, L ha-1). As soon as the culture reached the stages of R2, R3, R4 and R5, the cuttings of the whole plants were performed in the useful area of each plot and at the cutting height of 25 cm, in order to achieve maximum milk production per hectare, considering beyond the quality of the ensiled mass, also a larger amount of stored dry mass. The cut in the aforementioned reproductive stages, aim to represent the different harvesting points observed in the region of study, where most farmers, because they are family farmers, depend on the outsourcing of the service of harvest. In this way, some crops occur when maize is in the R2 stage (anticipating the harvest), or in R5, due to the high demand of farmers for this type of service. On the other hand, the producers with better implement structure, which can perform the harvesting when they deem appropriate, perform the cut normally when the plants are in R4. The samples were packaged in mini PVC silos with 75 mm in diameter and 50 cm in height. After 30 days of Data were subjected to analysis of variance by the F test using the statistical software sratgraphics (12.5). When the data did not present interactions among the factors (hybrids and reproductive stages of cutting), were analyzed as simple effects. The averages that showed significance were compared by the Tukey test (P < 0.05). .

III. RESULTS AND DISCUSSION
The interaction between the factors (hybrids and reproductive stages of cutting) was not significant (P < 0.05) in the variables studied, and the isolated effect of each factor was analyzed, as can be seen in table 2 and 3. For the hybrid maize factor, there was no significant effect for the variables MV, MS and PB. However, there was a difference (P < 0.05) between the response variables, starch, NDT, NDF and FDA (table 2). Observing the data presented in table 2, it is noted that the hybrids used in the experiment, because they are of double aptitude (grain and silage), have qualitative and quantitative characteristics within the expected standard for a good quality silage. As shown in Figure 1, one of the factors responsible for this result was the good climatic conditions, recorded during the development of the culture.
The production of 84.51 and 87.70 Mg of MV ha-1 in the hybrids analyzed were much higher, the production above 55 Mg of MV ha-1 considered by Neumann et al., (2015), as characteristic ideas of a good hybrids. This high production per hectare, in addition to allowing greater production of meat or milk per area, still dilutes the production costs. There was no difference between the crude protein values between the hybrids and neither between the different reproductive stages (table 2 and 4). COSTA et al., (2006) considers that PB contents below 7% could be limiting animal production, because they imply less voluntary consumption, reduction in digestibility and negative nitrogen balance. However, it is observed that the silages produced in this work (table 2 and 4) would satisfactorily meet (PB ≥ 7,81) to the minimum requirements of ruminants. On the other hand, in the case of milk production, the PB contents would be limiting the productivity, since the NDT values could provide conditions to obtain higher production values.
In the different reproductive stages of cutting, there was a significant statistical difference (P < 0.05) for the variables MV, DM, starch, NDT, FDN and FDA, with no difference between treatments only for the variable PB (table 3).  Cabral et al., (2012) The higher the proportion of grains in the mass, the higher the values of total digestible nutrients found. Therefore, besides the total mass production, it should also be considered the quality of the silage, which is influenced by the proportion of the plant components and the advancement of the reproductive stage of the plant. Thus, despite the increase in the fraction in more advanced stages of maturation, the increase in grain participation, provides higher energy levels and the quality of silage in greater proportion, than the losses caused by the fibrous portion (Oliveira, 2010), a situation observed in this work. The FDN and FDA values (table 3) decrease with the advancement of the reproductive stages, this result is due to the greater participation of grain in silage, consequently higher energy value (starch) and higher nutrient content digestible (NDT). Ensiled crops, before the ideal point, besides presenting lower total mass production, have a higher NDF content due to the occurrence of effluent and the lower share of the grain fraction in the mass ( VAN SOEST, 1994;VILELA et al. 2008). Similarly, Marafon et al. (2015), evaluating different maize harvesting stages, observed that maize plants in the R5 stage presented lower FDN and FDA content, when compared to the R3 stage due to the dilution of these fractions, by the transformation of simple sugars into starch in the grain component. Also, VAN SOEST et al., (1991), points out that good silage must comply with some minimum quality criteria.
Thus, values lower than 50% of neutral detergent fiber and 32% of acid detergent fiber (table 4), are important because they have a high correlation with the capacity of daily dry matter consumption and energy density of the resulting silage, respectively (VAN SOEST et al., (1991). For the production estimate (L cow-1 day-1; L ha-1), a significant isolated effect was verified between the hybrids and between the reproductive stages of cutting. Thus, the production estimate was higher for the hybrid P32R48 VYHR®, with values of 15.43 L cow-1 day-1, 16900.4 L ha-1, while the P30F53 VYHR®, obtained an average yield of 14, 47 L Vaca-1 day-1, 15845.7 L ha-1 (table 4). Averages followed by the same letter in the column do not differ from each other (P < 0.05) by the Tukey test. Standard error of Mean ± (0.299 L cow-1 day-1), and ± (327.69 L ha-1).
The highest production observed in hybrid P32R48 VYHR®, possibly due to the fact that it has a higher MS content, which raises the amount of PB available (table  2), approximately 90 kg of PB more per hectare, even if it does not differ from the other treatment. According to Pereira et al., (2007), the MS and PB contents of the plant are important factors in the ensilage process and in the determination of the nutritional value. This hypothesis may be considered because the protein in this case is the limiting factor of production, therefore greater availability of PB, higher production. In addition to being highly demanding in energy, lactating animals also present high protein demand that can limit the productive potential of these animals. The protein content in the diet has a positive correlation with the consumption, being this effect partly from the increase in   (table 7). In relation to the economic performance of the different reproductive stages, the R5 and R4 are superior to the other treatments, with no difference between the same (Table 7). Averages followed by the same letter in the column do not differ from each other (P < 0.05) by the Tukey test. Standard error of mean ± (631.918).
It can be seen that when the harvest of corn for whole plant silage was performed at the R2 stage, although it provides the producer with the removal of the early crop of the crop and allows to anticipate the sowing of the second crop, the producer fails to win, R$9353.11 Ha-1 when compared to the cut performed in R5. It is also important to emphasize that the ensilage process can be done between the R4 stages and R5 without significantly compromising the economic performance of the system, since the difference between these two cutting moments (R4 and R5) is only R$198.285 ha-1. This result is important as it allows a longer interval of time for the producer to perform the harvesting and ensiling of the material, decreasing the disorder and financial losses to the system.