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Bovine Tuberculosis - Analysis of 10-year cases and impact of visual inspection in the surveillance at the slaughterhouse in Portugal

Bovine Tuberculosis (bTB) is a One Health issue that interconnects human health with animal and environmental health. Due to its importance, cattle (the main host) it is under a national eradication programme in Portugal. Within it, meat inspection is considered a cost-effective procedure in bTB surveillance. The Commission's Implementing Regulation (EU) 2019/627 changed the post-mortem meat inspection requirements applied to cattle, essentially reducing handling and incisions in young cattle (<8 months). To study the impact of these changes on the surveillance of bovine Tuberculosis, data (age, sex, fitness, breed, lesions, laboratory results and post-mortem decisions) on suspected and confirmed cases of bTB detected at routine slaughter of the period from October 2010 to January 2020 were analysed. From a total of 3,279,548 slaughtered animals, meat inspection detected 808 suspected cases. Of these, 381 cases were laboratory confirmed. The estimated global prevalence was 11.6 cases/100,000, increasing with age: 3.5, 5.9 and 27.3 cases/100,000 in, respectively, the groups <8 months, 8 to 19 months, and ≥ 20 months. The prevalence was higher in females (16.3 vs 7.3 cases/100,000 in males) and in beef cattle (16.3 vs 1.4 cases/100,000 in dairy cattle). Among the confirmed cases, mediastinal and bronchial lymph nodes were the most reported locations. Based on the results, there is no evidence of a negative impact on the application of Regulation (EU) 2019/627 on bTB surveillance in animals ≥8 months. For the <8 months group the change to a visual inspection protocol could lead to a failure in the detection of bTB cases. Considering the epidemiological situation, the incision of the mediastinal, bronchial and retropharyngeal lymph nodes in cattle <8 months should be maintained.

Introduction
Bovine Tuberculosis (bTB) is a chronic zoonotic disease caused by bacteria from Mycobacterium tuberculosis Complex, mainly by Mycobacterium bovis [1]. Regarding public health concerns and the economic impact on animal production, in Portugal there is an eradication programme (PETB) since 1991 aimed to control the infection in susceptible animal hosts, thus reducing the risk for human infection [2][3][4].
Under the PETB, at the farm level, the screening for bTB is based on the single intradermal comparative tuberculin skin test (CITT), which is considered the most widely used test [5][6][7][8]. After a positive test, the animal is slaughtered in the abattoir under special conditions, a sanitary slaughter. A detailed post-mortem inspection is performed to identify granulomatous lesions compatible with bTB infection [3] as a supplementary means of monitoring the effectiveness of surveillance screening tests [9,10]. The gamma-interferon test can also be used as a complementary diagnostic test, when there is a significant number of doubtful results in CITT and as an alternative to compulsory sanitary slaughter on herds with chronic positivity on CITT [3,6].
Meat inspection is essential to verify compliance with requirements for human and animal health and animal welfare and it is also a valuable tool in animal health surveillance and monitoring [11][12][13]. With this perspective, meat inspection plays a primary role in detecting animals that despite being CITT negative, do present macroscopic lesions at postmortem examination, being considered a cost-effective procedure in bTB surveillance [7,8,10,[14][15][16].
Practical arrangements for post-mortem inspection are defined in the Commission Implementing Regulation (EU) 2019/627, which introduced changes in official controls moving forward to risk-based meat inspection [17]. Particularly in young cattle (<8 months), if there are no indications of possible risk for public health, animal health or welfare, palpations and incisions are reduced to minimize cross contaminations. The main procedures that were suppressed included the incisions of the retropharyngeal, bronchial and mediastinal lymph nodes, and may have repercussions on the detection of granulomatous lesions of bTB, since they occur frequently in those locations [18]. A change in these procedures under a routine slaughter may be associated with a reduction in the likelihood of detecting lesions consistent with bTB and, consequently, may have a negative impact on disease control programmes and public health [19,20]. To ensure the surveillance of their tuberculosisfree status Member States may decide to carry out further investigations [11] Therefore, each EU member state should assess the possible consequences of the implementation of this new inspection protocol on bTB surveillance.
The objectives of this study were to characterize the cases of bTB detected through post-mortem inspection at the abattoir over a 10-year period and to investigate the possible impact of the change in the meat inspection protocol on the surveillance of the disease.

Data collection and case definition
The study population, the total of cattle slaughtered in Portugal from October 2010 to January 2020, was obtained from the Animal Identification and Registration National Database (SNIRA). The National Competent Veterinary Authority Official Database for meat inspection (SIPACE) was consulted to collect data on the suspected and confirmed cases of bTB detected during routine slaughter from October 2010 to January 2020 in all Portuguese bovine slaughterhouses. These cases correspond to animals that had tested negative for CITT but presented detectable macroscopic lesions on post-mortem examination at slaughter. A suspect case was defined as an animal slaughtered for human consumption that in the post-mortem examination performed by the official veterinarian (OV), presented one or more macroscopic lesions compatible with bTB. According to the national guidelines, all suspected lesions of bTB detected in animals originating from officially bTB-free herds, and those not officially free but without isolation of bacteria of the Mycobacterium tuberculosis complex, are collected by the OV and examined for histopathologic and/or bacteriological confirmation at the national Reference Laboratory [3]. The confirmed cases of bTB are the result of the bacteriological isolation of any bacteria of the Mycobacterium tuberculosis complex and/or compatible histological examination, as defined in the PETB [3,4].
The data collection of suspected and confirmed cases included animal age, sex, breed, type of production, lesions distribution, laboratory results and post-mortem decision. When some information was missing, other official databases were consulted, such as the national animal identification and registry database SNIRA. Despite these efforts, in some cases data were missing, namely the location of lesions.
The carcasses were declared fit or unfit for human consumption by the Official Veterinarian, according to EU law [11].
Following the age categorization of the Commission Implementing Regulation (EU) 2019/627, of 15 March 2019, animals were divided into three classes: <8 months, 8 to 19 months, and ≥ 20 months. The type of production was categorized as beef cattle and dairy cattle and subdivided into two other groups, autochthonous (Portuguese) and exotic breeds.
The location of lesions included lungs, pleura, and lymph nodes (retropharyngeal, parotid, mandibular, mediastinal, bronchial, gastric, mesenteric, hepatic, renal and retro mammary), spleen, liver, kidneys, peritoneum, genitals, mammary gland. The assessment of the location of lesions was carried out in the slaughterhouse and registered in accordance with the official guidelines [3] In this study, lesions were categorized as single, if only one organ or lymph node was affected, and multiple if else.

Data analysis
The prevalence of abattoir cases of bTB was calculated by dividing the number of confirmed cases by the population and multiplying by 100,000. The prevalence was reported as cases per 100,000. The confirmation rate was calculated by dividing the number of confirmed positive cases by the number of suspected cases of bTB and was reported as a percentage. Absolute frequencies and percentages were used to describe the distribution of suspected and confirmed cases of bTB. The distribution of lesions in bovines suspected and infected was assessed. Age in months was described as median and the interquartile range (IQR).
The data were analysed using Excel and SPSS version 26.

Results
A total of 3,279,548 animals were slaughtered from October 2010 to January 2020, 808 animals were classified as suspected cases, and of these 381 (47.2%) were confirmed by laboratory analysis (Table 1). Mycobacterium bovis was isolated in 80.3% (n = 306) of the positive cases; Mycobacterium caprae in 1,8% (n = 7), Mycobacterium tuberculosis in 0,3% (n = 1) and Mycobacterium tuberculosis complex in 1,3% (n = 5). The remaining 13,6% were divided between compatible with bTB by histopathological examination (6%) and positive results registered in the database without any information about laboratory results. The confirmation rate and the number of confirmed cases reached the highest values in 2018 and 2019, respectively 79.1% (n = 53) and 71.1% (n = 59). The estimated prevalence of bTB over the ten-year period showed an increasing trend from 2015 to 2020. The estimated global 10year prevalence was 11.6 cases/100,000, increasing with age: 3.5, 5.9 and 27.3 cases/100,000, respectively, in the groups <8 months, 8 to 19 months, and ≥ 20 months ( Table 2). The prevalence was higher in females (16.3 vs 7.3 cases/100,000 in males) and in beef cattle (16.4 vs 1.4 cases/100,000 in dairy cattle). All dairy animals were of exotic breeds, while beef cattle included both exotic and autochthonous breeds. Among beef cattle, autochthonous breeds account for only 14% of slaughtered animals, but the estimated prevalence of bTB in these was higher than in exotic breeds (32.2 vs 13.9 cases/100,000).
The median age of the confirmed cases was 56 months (interquartile range: 16 to 137 months), higher in females (median = 110 and interquartile range: 34 to 161 months) than in males (median = 17 and interquartile range: 12 to 38 months), and higher in beef cattle (median = 61 and interquartile range: 16 to 140 months) than in dairy cattle (median = 25 and interquartile range: 12 to 50.5 months).
Regarding the distribution of lesions, of a total of 799 suspected cases with information, 432 (54.1%) had multiple lesions and the rest had single lesions. Detailed information on the location of lesions (Table 3) was only available in 767 records. The most reported location in suspected cases was the mediastinal lymph nodes (46.3%), followed by the lungs (46%) and bronchial lymph nodes (37.5%). In the confirmed cases of bTB, the locations of the lesions were the mediastinal (62.5%) and bronchial lymph nodes (53.9%), the lungs (49.2%), and retropharyngeal lymph nodes (33.1%). Considering the three age groups in the confirmed cases, mediastinal and bronchial lymph nodes were also the most reported locations. However, retropharyngeal lymph nodes were the third most reported location in the two youngest groups, versus lungs in the oldest group. Considering the anatomical regions, in the confirmed cases the highest proportion of lesions was in the thoracic region (83.9%) and the head (34.7%).
Analysing the occurrence of single lesions in confirmed cases (n = 109), only 107 had information on the anatomical location (Table 4). In these cases, retropharyngeal lymph nodes (44.9%), followed by mediastinal lymph nodes (27.1%) were the most common locations of lesions. The anatomical distribution of single lesions across the three age groups was very similar and in accordance with the distribution of the total number of cases. In the confirmed cases, the total condemnation rate of carcasses was higher (57.6%) than in suspected cases (45.2%).

Discussion
A post-mortem examination at the slaughterhouse constitutes an additional surveillance method for detecting cases that may have been missed in ante-mortem testing programmes [10,15,[21][22][23]. In our study, nearly half (47.2%) of the suspected cases detected in the slaughter line were confirmed as positive cases, a lower proportion than reported by other authors [24,25]. Mycobacterium bovis was isolated in 80.3% (n = 306) of the positive cases, in accordance with the sensitivity of the bacteriology testing previously described [26]. Surprisingly, since the PETB is in force for a period of 31 years, it was observed an increasing trend in the detection of cases since 2015 reaching the highest value in 2019 ( Table 1). As meat inspection procedures were maintained in the reporting period, this may reflect the increased sensitivity of the OV to detect lesions through post-mortem examination, encouraged by the Competent Authority under the PETB. During the last four years of the period under analysis, the confirmation rate has also increased. This can indicate an increase in the submission to the laboratory of granulomatous lesions with a macroscopic appearance clearly indicative of bTB, improving the accuracy in the laboratory results.
The prevalence was higher in beef cattle and within these, in autochthonous breeds. These animals are raised in a more traditional way, preferably outdoors with access to pasture that can potentially lead to direct or indirect pathogen transmission at the wildlife-livestock interface in a multi-host system [27][28][29][30]. In our study, older animals (≥ 20 months) and females (median age 110 months) had the highest proportion of confirmed cases. Our results are in accordance with previous studies, regarding the fact that for sake of production and economic interests, cows usually reach an older age than males because of their role in calving and the increase in incidence with age reflects an intrinsic property of bTB transmission [29,31,32].
In confirmed cases, the most recorded locations of lesions (Table 3) support the prevalence of respiratory infection and are in agreement with previous studies [18,[33][34][35] Interestingly, 71.2% of the confirmed cases (279 out of 378) presented multiple lesions in opposition to other studies, that reported above 70% of single lesions [36,37]. Whenever a granulomatous lesion is detected, the OV performs a more detailed examination with additional palpations and incisions, increasing the probability of finding multiple lesions [11].
Several authors and studies confirm that post-mortem inspection including palpation and incision is more effective to detect bTB lesions when compared with the visual inspection method [13,15,24,25,38,39] can be hypothesized that the use of a simplified inspection method decreases the chance to find gross lesions and can delay the success of an eradication program [10,25]. However, there is a trend in the modernization of meat inspection in the EU food law moving forward to risk-based meat inspection, avoiding unnecessary manipulation and consequent cross-contamination [12,17].
A qualitative risk assessment of the visual-only meat inspection concluded that Mycobacterium bovis was the only risk confirmed as nonnegligible for animal health and welfare, since the omission of palpation and incision of lymph nodes and organs is likely to reduce the sensitivity of detection of bTB-associated lesions on post-mortem examination to almost zero [38]. The risk to animal health was considered higher in regions or countries where bTB was officially eradicated, given the Table 1 Number and distribution of suspected cases (N = 808) and confirmed cases (N = 381) of bovine tuberculosis per year in the period 2010-2020 and estimated annual prevalences.  longer time interval between CITT controls [38]. The main changes introduced by the Regulation (EU) 2019/627 regarding post-mortem inspection for the detection of bTB are, in bovine ≥8 months, the suppression of visual inspection and of incision of the sub-maxillary and parotid lymph nodes and the lungs. In our study, out of 344 animals ≥8 months, only one case with a single lesion in the submaxillary lymph node and six cases with lesions in the lungs were identified. In these latter six cases, either a complete description of the location of the lesions was missing in the data base or the respective lymph nodes were not carefully examined, since additional lesions should be expected. This can be explained by the pathogenesis of tuberculosis. A primary complex is complete if lesions in the primary focus of infection and respective draining lymph nodes are both present, and incomplete, i.e., with single lesions in the lymph nodes, when the lesion at the portal of entry has already disappeared [14].
In young bovine animals <8 months, the meat inspection protocol was deeply altered by Regulation (EU) 2019/627. Post-mortem inspection became mostly visual, maintaining only the palpation of lungs, retropharyngeal, mediastinal and bronchial lymph nodes and suppressing all incisions. In this age group, except for one case with generalized lesions, we found that all cases presented lesions of early stages of infection -primary complexes that could be unnoticed with a visual-only post-mortem examination. Single lesions were found in 7 out of 18 confirmed cases and these were located in the retropharyngeal or mediastinal lymph nodes. In the retropharyngeal lymph nodes, 80% of the confirmed cases had single lesions and, in the mediastinal lymph nodes, the opposite was found, revealing multiple lesions in 75% of the cases. However, most of these multiple lesions (78%) were associated to primary complexes. Despite the estimated prevalence of bTB being low in this group (3.5 cases/100.000), the importance of early detection of bTB outbreaks should not be neglected, avoiding the dissemination and increase of the infected herds. In a future study, it would be useful to compare the results of meat inspection on the detection of new bTB outbreaks with the testing in live animals through CITT. This practice would be a good tool to evaluate the contribution of post-mortem inspection to the detection of bTB cases.

Conclusion
To our knowledge, this is the first study with a detailed analysis of the cases of bTB detected in slaughtered cattle in Portugal. This zoonotic disease is a global veterinary public health issue, which clearly justifies the One Health approach. In Portugal, due to its importance, it is under an eradication program (PETB) in cattle, the main reservoir. The results from this study highlighted that post-mortem inspection of routinely slaughtered cattle have proven to be a valuable tool for screening bTBinfected animals that for some reason were not detected at the farm level under the PETB. The variety in the typology and topography of lesions found in bTB-confirmed animals, allowed us to conclude that there was no evidence of a negative impact on the surveillance in cattle ≥8 months old by changing post-mortem inspection protocol to implementing Regulation (EU) 2019/627. On the contrary, for the group <8 months of age, despite the limited number of cases confirmed (n = 18, 3.5 cases /  100,000) our data suggest that changing the procedures to a visual-only inspection protocol, could lead to a reduced detection of bTB cases, resulting in a detrimental effect in public and animal health. Based on this Regulation, incisions may be maintained considering the epidemiological situation of the Member State. The results of our study can provide scientific support to the national Competent Veterinary Authority's decision to maintain the incision of mediastinal, bronchial and retropharyngeal lymph nodes in bovine animals <8 months.

Funding information
This work was financed by National Funds (FCT/MCTES, Fundação para a Ciência e a Tecnologia and Ministério da Ciência, Tecnologia e Ensino Superior) under the project UIDB/00211/2020.
The participation of Madalena Vieira-Pinto was supported by the projects UIDB/CVT/00772/2020 and LA/P/0059/2020 funded by the Portuguese Foundation for Science and Technology (FCT).


provenance: AgriculturalAndFoodSciences,2022-2022-Agricultural And Food Sciences-2022.jsonl.gz.gz

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A suite of agronomic factors can offset the effects of climate variability on rainfed maize production in Kenya

Achieving food security in sub-Saharan Africa (SSA) is a multidimensional challenge. SSA reliance on food imports is expected to grow in the coming decades to meet the population's demand, projected to double to over 2 billion people by 2050. In addition, climate change is already affecting food production and supply chains across the region. Addressing these multiple food security challenges will necessitate rapid enhancements in agricultural productivity, which is influenced by a host of demographic, agronomic, and climatic factors. We use statistical approaches to examine rainfed maize in Kenya, where maize cultivation and consumption are widespread and central to livelihoods and national food security. We find that improving a suite of agronomic factors, such as applying fertilizer, planting certified seeds, and extension services, will have a greater effect on rainfed maize productivity than demographics and can offset the effects of climate change. These findings could also offer insights into similar challenges for other crops in Kenya and other SSA countries.

www.nature.com/scientificreports/ services) 13,15,17 . Other work on smallholder demographics found that educated farmers have better uptake of improved technologies 13,15 . All this previous work indicates that the demographics of smallholder farmers are essential to consider in developing effective strategies to improve food production in SSA. Climate variability and change continue to disrupt rainfed agriculture in SSA primarily through changing rainfall patterns, rising annual temperatures, and increasing extreme events 18 . Extensive research has examined the relationship between climatic factors and rainfed yield outcomes in SSA. While climate change-induced changes in SSA's suitability for maize (Zea mays) production will vary by agro-ecological zone, the overarching trend across agro-ecological zones indicates declining yields 19 . Rainfed production of cereals in SSA is projected to increase due to the use of improved technologies, but potentially attainable yields are likely to be reduced due to changing climatic conditions 20 . Heavier rain will also increase nitrogen leaching, leading to reduced plant uptake and lower yields in nitrogen-deficient -soils 21 . Other studies focused on the effects of temperature have generally found a negative relationship between temperatures and crop productivity 3,22,23 . However, rising temperatures may increase yields in certain regions (i.e., the Ethiopian highlands and the continent's southern region) 22 . Smallholder farmers in SSA are some of the most vulnerable to the impacts of climate variability and change and identifying opportunities to increase their yields can improve their adaptive capacity 24,25 .
Sub-optimal agronomic factors have led to lower yields compared to the attainable rain-limited crop yield 10,13,26 . This yield gap between actual and attainable crop yields is estimated to be up to 80% for certain crops in SSA countries 27 . This under-productivity is primarily the result of low uptake of improved inputs such as certified seeds and fertilizer 12,15,28,29 , a lack of economic incentives, or services, and insufficient capital for smallholder farmers 25 . Further, improved farm equipment are often designed with large farms in mind, unsuitable for hilly and stony smallholder farms, and uneconomical except through rental schemes or farmers' associations; as a result, their per capita ownership is also low in SSA 30,42 . Improving physical and economic access to these inputs and services is another vital component for enhancing smallholder productivity.
As evidenced above, a large body of work has sought to understand the relationship between smallholder yields and demographic, agronomic, or climatic factors in isolation. However, little work has evaluated the relative importance of these three sets of factors together in ultimately determining rainfed yield outcomes. Here we explore this knowledge gap by examining the case of rainfed maize in Kenya, where maize cultivation and consumption is widespread, central to farmer livelihoods, and essential for national food security. We leverage detailed, nationally representative farmer survey data for 2010 and 2013 to evaluate the relative importance of demographic, agronomic, and climatic factors in influencing maize yields. By examining the relative effect of these factors, this study aims to identify the factors that offer the greatest opportunity for improvements of rainfed maize yields in Kenya, to understand whether factors under farmers' control can overcome the effects of climate variability on yields, and to draw broader inferences that are generalizable to similar challenges in rainfed production of other crops in Kenya and in other SSA countries.

Results
We examined the significance and effect-size of each predictor variable within our three groupings: farmer's demographic information, agronomic factors, and climatic conditions ( Fig. 1 and supplementary Table 3).
Model coefficients for the farmer demographic variables had relatively small magnitudes, with farmer education having a significant (p = 0.0293) and modest relationship to maize yield. We find that one standard deviation www.nature.com/scientificreports/ increase in farmer's education corresponded to a 0.049 unit increase in normalized maize yield. Gender, household size, age, and farming experience showed no statistically significant association with maize yield. Farmer agronomic factors had the highest number of variables with a significant relationship with normalized maize yield. Farmers who used fertilizer had a 0.362 unit increase in normalized maize yield, while those who planted certified maize seeds were associated with 0.197 units higher productivity than non-certified maize seeds users. For every standard deviation increase in the maize plot size, normalized maize yield was reduced by 0.046-a confirmation of the inverse field size-yield relationship 12,16 . As the normalized time to the nearest market increased by a single unit, the normalized maize yield decreased slightly (− 0.048). At the same time, farmers who had accessed extension services in the current or a previous season had increases of 0.116 and 0.195, respectively, in normalized maize yield units. Access to credit, and distance to (government) extension services did not significantly correlate with normalized maize yield (other factors, such as the use of certified seeds and the application of fertilizer, through which access to credit and distance to extension services may impact maize yields may be influencing their effect-size and statistical significance).
Climatic conditions showed significance in the associations of maximum temperature and normalized maize yield. The linear term of maximum temperature had the largest effect-size of 1.727. However, the squared maximum temperature term had a negative effect of − 1.712, suggesting that the change we observed in normalized maize yield due to maximum temperature has an optimal value beyond which temperature begins to adversely impact yields. One standard deviation increase in annual precipitation linear term had a positive effect of 0.161, and the squared annual precipitation term had a marginally significant coefficient of 0.135.
To verify our results, we created models for each agroecological zone. Overall, the estimated coefficients were consistent in size across all agroecological zones except the climatic variables, and the combined effect of agronomic factors had the most significant impact on normalized maize yield, followed by climatic conditions and, lastly, farmer demographics.

Discussion
Our analysis provides new understanding of the relative importance of demographic, agronomic, and climatic factors in influencing maize yields in Kenya and provides valuable insights into the ways in which these factors may combine to determine yield outcomes for other crops and other countries. We find that agronomic factors have a relatively high influence on yield compared to farmer demographics and offer the greatest opportunity for improvements of rainfed maize yields and counteracting the effects of climatic factors. No single agronomic factor has an effect-size large enough to offset the effects of climatic conditions, and adoption of agronomic factors can be correlated-use of fertilizer and certified seed is an example (Table 1). Thus, a suite of agronomic factors is necessary to improve smallholder yields while adapting to climate change (Fig. 2).
We show that farmers who plant certified seeds, apply fertilizer, or access extension services register higher maize yields; however, due to lack of data, we did not study how the maize yield would vary depending on the type of certified seed planted, how the farmer applied fertilizer, or what extension services they accessed. Efforts www.nature.com/scientificreports/ aiming to increase yields to feed the growing SSA population and mitigate the effects of climate variability and change should focus on the factors that can most enhance productivity. By comparing the relative effect sizes of each demographic, agronomic, and climatic factor, we show that the mean relative effect-size of agronomic factors is less than that of climatic factors, providing evidence that a suite of agronomic factors is needed to offset the effects of climate variability, consistent with other studies 10,31-35 . Unlike climatic factors over which farmers have no control, agronomic factors are the set of factors most at agency of SSA farmers. Consequently, efforts to close maize yield gaps-and by extension, the yield gaps for other staple crops-should prioritize targeted improvements in agronomic factors. In particular, our findings provide evidence that faster access to markets, providing extension services, planting certified seeds and applying fertilizer can have an immediate and positive effect on yield outcomes. We also confirm that smallholder farmers with smaller fields tend to be more productive than those with larger fields, as smallholder farmer's precision in implementing agronomic factors relates inversely to farm size 16,30 . We note though that our findings do not downplay the effect of demographics in potentially improving yields. We find that farmers with higher levels of education tend to have better yield outcomes, which can potentially be attributed to their increased awareness of farming factors and market dynamics 15 . Other farmer demographics, such as gender, have been shown elsewhere to affect yield 17 despite not having a significant relationship with yield in this study.
While further research is needed to understand interactions of demographic, agronomic, and climatic factors in determining smallholder yields, our study points to multiple opportunities for holistic approaches to improve farmer productivity and to offset adverse impacts of climate change and variability. While our findings are based on rainfed maize cultivation in Kenya, we provide a generalizable methodology and set of results that can be applied to other crops and countries in SSA. Though smallholder options for improved productivity differ based on the crop of interest, government policies, agroecological environments, and climatic conditions, our findings indicate that improved agronomic factors can play an important role in addressing the overarching challenges of population growth, yield gaps, and changing climate.

Methods
We evaluated the sensitivity of rainfed maize yields in Kenya to demographic, agronomic, and climatic factors using a linear model. We developed the linear model with farmer survey data collected in two calendar years (2010, 2013) covering six agro-ecological zones and thirty-two counties.

Study area. The study focused on the main maize growing areas in Kenya. Kenya is in East Africa and has
forty-seven counties (i.e., level-one administrative units). As of 2010, The forty-seven counties were districts under the Kenyan national government. After the March 2013 general elections, the districts' restructuring into counties was completed in line with a devolved system of government outlined in a new Kenya constitution passed in 2010 (for consistency, the word counties is used even for 2010). The study covered thirty-two counties across the southern part of Kenya. In 2010, these thirty-two counties accounted for 91% of total maize production (Fig. 3) and covered 92% of the total area under rainfed maize production 36 . Survey: data collection, processing, and cleaning. The International Maize and Wheat Improvement Centre (CIMMYT), in collaboration with the Kenya Agricultural and Livestock Research Organization (KALRO), conducted nationally representative household surveys in the major maize growing areas. The data and the surveys have been previously used to analyze trends in mechanization 37 and fertilizer use 33 .
The 2010 and 2013 surveys used a two-stage stratified design with six maize agro-ecological zones (AEZs) as strata 31 , census clusters or sublocations as primary sampling units, and maize growing households as secondary sampling units. The first survey, done in 2010, covered 120 sublocations with 1344 households. The second survey, done in 2013, interviewed the same farmers with a 20% replacement with randomly sampled households 34 .
CIMMYT-Nairobi and KALRO surveyed the farmers per the guidelines of the Declaration of Helsinki, and the data was provided for this study by CIMMYT-Nairobi, to the authors, after signing a confidentiality agreement. The farmers provided all information in the survey after being taken through and signing a consent form.  Tables 1 and 2). The 17 variables include one target variable (maize yield in kg ha −1 ) and sixteen independent variables. We organized the independent variables into three broad groups: farmer's demographic information, agronomic factors, and climatic conditions. There were six variables on farmer demographics: gender, marital status, age, size of household, years in farming, and years of education; eight variables on agronomic factors: area under maize, use of certified maize seeds, use of fertilizer, access to extension services (in current and previous seasons), distance to the nearest extension service center, access to credit services, and, time to the nearest market; and two on climatic conditions (i.e., variables largely out of a farmer's control): maximum temperature (at 2 m height), and total precipitation in the growing season. The original dataset also included a minimum temperature variable, www.nature.com/scientificreports/ which we excluded from the analysis, as it provided near-identical information to maximum temperature in explaining yield variability. We mapped the spatial distributions of the independent variables to identify spatial trends in the data (Supplementary Figs. 1-30). We transformed each independent variable to the appropriate data structure before using them in the linear model. First, we added the squared terms of the climate variables (maximum temperature and precipitation) as new variables because of the non-linear relationship between climate and yield. Then, we set all binary variables (Table 2) as dummy variables with their absence as the reference value, set marital status as a categorical variable of three levels, and normalized the numeric variables. We normalized numeric variables to remove the effects of measurement units in two steps: subtracting the mean from each observation and dividing this difference by the standard deviation. Before normalizing, the climate variables were divided into groups based on the agroecological zones, and each group normalized separately. Lastly, we calculated the variance inflation factor (VIF) to eliminate colinear independent variables based on a standard VIF threshold of five. The VIF value of all variables was less than the threshold, so we did not remove any.
Linear model. Using the normalized data, we developed a linear model to examine how sensitive maize yield (kg ha −1 ) was to farmers' demographic information, agronomic factors, and climatic conditions following 38 and 39 . We computed the linear model and performed the data normalization and VIF calculation steps described in "Survey: data collection, processing, and cleaning" section in R using functions from stats, fmsb, and R base packages. We organized the developed functions as a new R package and anonymized the data used in this study before including it in the R package, which we called "yieldest. "The R package is publicly available on GitHub 40 and Harvard Dataverse 41 . Using normalized maize yield as the target variable and the normalized independent variables, we could compare the magnitude of coefficients (i.e., effect size) in the linear model to evaluate the relative influence of corresponding predictor variables in determining rainfed maize yields.

Equipment and settings.
We generated all figures in this manuscript using ggplot2 package R version 4.1.2 (Rstudio version 2022.02.0 + 443 in windows 10) with data from our results and gridded maize yield data (the year 2010) from 35 .

Data availability
The datasets generated and/or analyzed during the current study are available in the Harvard Dataverse repository, https:// doi. org/ 10. 7910/ DVN/ UIWQQH. Contact corresponding author for data requests.


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Effects of Selected Pigments on the Properties of Silicone Resin-Based Paints

The purpose of this research was to evaluate the impact of selected pigments on the performance of waterborne emulsion paint. Each pigment was incorporated into the paint at 5% w/w. Density and viscosity measurements as well as the rub-out test were used to test the wet state properties of the colored paint. Wet-scrub, adhesion-to-substrate, water-uptake, vapor-permeability, UV-aging, and other tests were conducted to evaluate effects of the pigments on the dried paint. Bohemian green earth pigment was found to have the most positive effect, as it improved the water resistance of the paint without changing its rheological properties. Therefore, this pigment was selected for further studies, in which the pigment was included as part of the paint formula rather than as a post-additive. The results were satisfactory, confirming the compatibility of the pigment with the formula. However, a slight change in the rheological profile of the paint was observed during tests on a rotational rheometer. This research shows the need for intensive quality control measures while testing alternative formulations, to both enable early detection of negative effects and identify possible improvements.

Introduction
Waterborne emulsion paints are widely used as exterior coatings in the construction industry. The properties and applications of waterborne emulsion paints have been well documented [1][2][3][4]. They show excellent performance when it comes to durability, water resistance, and flexibility. They are also simple, single-component products that are ready to use. However, the properties of waterborne emulsion paints continue to be improved [5][6][7][8].
Most manufacturers now offer waterborne emulsion paints in a wide range of colors. Colorants are usually added in the form of pigment pastes instead of dry pigments. This method ensures that the pigments have a minimal impact on the coating properties, provided they are not used excessively. However, such interference does not necessarily diminish the properties of the paint. It can maintain or even improve its performance in some areas. There are many different types of colorants, which affect more than just the color of the paint [9][10][11].
Most research focuses only on the properties that are supposed to be changed by the pigment (or other additive). Some studies also consider the ability of cool pigments to reduce surface temperature [12,13], the properties of organic pigments [14][15][16] and spinel pigments [17][18][19], color changes of thermochromic pigments under temperature [20][21][22], or color fading [23]. However, there are few studies testing the impact of pigments on the functional and processing properties of waterborne emulsion paints or coatings [24][25][26]. Mentioned studies provide little research on the overall impact that additives can have on the product. This work presents the impacts and possible benefits of including dry pigments in coating formulas by testing modified paint for all important application and processing properties. These effects and slight improvements are not usually investigated while testing the properties of new pigments. In the first part of the research, several pigments (green earth, spinel, iron oxide) were added to a premade base of acrylic-silicone paint. Following tests on the liquid paint and hardened coating, one pigment was selected as the most suitable, and a new paint was formulated with the pigment included as a core ingredient. Subsequently, further tests were conducted to determine the properties of the new paint.
Pigments for tests were chosen from different groups, with various reasons to use them in paint. Iron oxide red is a well-known, UV-stable red pigment; green earths are chosen as more economical alternatives to popular cobalt greens; and zinc-iron bronze is a spinel-type pigment (iron and zinc oxide mineral), with potential to have additional positive effects on the product. Green earths have been used as pigments for thousands years [27]. They offer a less expensive alternative to other green pigments. The most popular green pigments-cobalt greens-are among most expensive, reaching prices of €150-200 per kg. Possible alternatives such as Nicosia Green can be purchased for €16 per kg [28]. Spinels have been shown to have beneficial effects in different applications; for example, reducing metallical defects in ceramics [29], or in anti-corrosive, heat-resistant coatings for metal applications [30]. In the present work, it has been shown that Bohemian Green Earth pigment may be successfully used as an effective coloring additive for paint formulation, while it had no major impact on the characteristics of the original paint, including its viscosity, wet-scrub resistance, and rheological properties. This work highlights the importance of extended product testing after any modification, as even minor changes in recipe can lead to differences in product properties.

Raw Materials
A standard waterborne acrylic-silicone facade paint (Atlas Sp. Z o. o., Łódź, Poland) was used as a carrier for the tested pigments. The following pigments were chosen for testing: The following commercial pigment pastes were used only as a reference for color aging tests (they were not taken into consideration when testing properties):

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Commercial green GX11-a pigment paste based on cobalt green pigment Co 2 TiO 4 . PG50-a spinel pigment obtained under high temperature from cobalt (II) oxide and titanium (IV) oxide. • Commercial red RX10-iron red, PR101, in the form of a pigment paste. • Commercial red RH6-organic red pigment, known by the color index PR254.
The following material was used as reference filler. It was added to the reference sample to maintain equal dry component concentration through the samples: o., Piotrowice, Poland)-standard dolomite filler used in the tested paint.

Sample Preparation
Samples were prepared by the addition of 5% w/w of each pigment to the base paint. The pigments were gradually incorporated to the samples using a rotary agitator at a speed of 1000 rpm. Each sample was placed in a sealed container until needed for research. P60 is a neutral paint filler used in the reference sample to equalize the pigment concentration with the other samples.

Test Methods
The following test methods were applied to determine the effects of each pigment on the material:

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Density measurements were performed using a density cup with a volume of 100.088 cm 3 to measure the weight of each sample, followed by density calculations according to the formula given in PN-EN ISO 2811-1:2012 [31]: where m is sample mass and V is volume. Each sample was measured three times. The rub-out test was performed by gently rubbing the surface of the applied paint with a fingertip after short time periods (1, 5, and 10 min after application) [34]. The rub-out test was used to analyze the compatibility of the pigment with the material by observing the occurrence of flocculation.

•
The wet-scrub resistance test was performed for 200 cycles of scrub on an Elcometer 1720 (Manchester, UK) using a sponge and a 5% solution of sodium dodecylbenzenesulfonate (ABSNa) as a wetting agent. The samples were prepared by the application of a 300 µm thick coating of each paint on a dedicated scrub test substrate. The coatings were conditioned for 28 days at room temperature to ensure complete drying. The dry samples were measured using a thickness meter and placed in the scrub resistance tester. After the test, the samples were rinsed with water and placed in an air dryer at temperature of 50 • C for 7 days. Subsequently, the thicknesses of the coatings was measured again. Scrub resistance class was determined based on loss of coating thickness (PN-EN 13300:2002 standard) [35]. • Adhesion to the substrate was measured using the TQC cross-cut adhesion test (Hadley, UK). Samples were applied to cardboard using a 300-µm applicator and left for 28 days at room temperature to dry out. Next, the surface of each sample was cut vertically and horizontally, with an angle of 90 • between cuts. Adhesion to the substrate was determined based on chipping at the incision edges. • Water absorption tests were conducted according to the PN-C-81521:1976 standard. Two equal layers of paint were applied to a limestone substrate with a 24-h wait between layers. The dimensions of the limestone substrate were 11.5 × 11 × 2.5 cm. The samples were left to dry out for 28 days at room temperature. The edges and sides of the limestone were covered with silicone to seal any areas not covered with paint. Two samples were prepared for each pigment. The samples were conditioned by dipping in water for 72 h, followed by drying in an air dryer in 50 • C for 72 h and resting for 24 h in room temperature. The samples were then weighed and placed in a container filled with water up to half the height of the sides of the samples. The samples were placed on a piece of sponge with the surface covered with paint facing down, to ensure contact with water throughout the test. After 24 h, the samples were weighed again and left to dry at 50 • C for another 24 h. The process was repeated three times. The water uptake factor was calculated according to the formula [36].
where W 24 is the water absorbance factor after 24 h, m 24 is the sample mass after 24 h in water, m 0 is the sample mass before dipping in water and S is the surface of the sample covered with paint.

•
Vapor permeability was measured using the wet-cup method, according to PN-EN ISO 7783:2018-11 [37]. Samples were prepared by applying two equal layers of paint on an oval substrate, with a 24-h wait between layers. The samples were left to dry out for 28 days. Measuring cups were filled with a saturated solution of anhydrous ammonium dihydrogen phosphate, leaving space of about 30 mm between the surface of the solution and the sample. The samples were placed on top of the measuring cups, sealed, and weighed. The edges of each sample were sealed with rubber bands to ensure vapor circulation exclusively through the sample. The cups were placed in a chamber with forced air circulation and weighed every 24 h for 4 days. The linearity of the graph shows that the test has been correctly executed.

•
Open time measurements were conducted using a Q-Lab open time tester (Westlake, OH, USA) equipped with a rod for scratching the surface of the sample and a timescale alongside the sample. As the scratching rod moves steadily over the surface, the timescale makes it possible to monitor the time passing. Coatings were applied with a 200-µm applicator on a glass plate. The measurements lasted for 15 min, during which the scratching rod moved through the sample to the point where it no longer damaged the forming film. Each sample was measured separately immediately after application [34].

•
Contrast measurements (mating) were conducted using an X-Rite Color i5 (Grand Rapids, MI, USA) spectrophotometer according to PN-EN ISO 2814:2006 [38]. Samples were prepared by applying a 300-µm film of paint on black-white contrast cards. The samples were conditioned for 28 days at room temperature. Mating ability was determined by measuring Lab values over white and black fields and calculating the ∆E values. • UV color stability was measured using a X-Rite Color i5 (Grand Rapids, MI, USA) spectrophotometer. The lab values were compared with reference samples using ∆E, according to PN-EN ISO 16474-3:2014_02 [39]. The samples were conditioned using a Q-Lab xenotest chamber (Westlake, OH, USA), where they were exposed to xenon arc lamps imitating full spectrum solar irradiance for 28 days at room temperature. Two series of measurements were conducted: ageing for 150 h and aging for 500 h. After that time, the lab values of the samples were measured • Particle size measurements were conducted using the light scattering method with a Zetasizer device (Malvern, UK). Samples were prepared by mixing 0.1 g of the pigment in 100 g of water. Ultrasound was used to maximize pigment dispersal. The samples were placed in measuring cuvettes and transferred to the Zetasizer device. Figure 1 shows the effects of the studied pigments on the density and viscosity of the final products. Viscosity is a main factor in determining the application properties of paints. Waterborne paints are designed to have specific rheological properties, such as sagging resistance or good leveling on walls [40]. It is important to maintain viscosity while changing ingredients. As can be seen in Figure 1, all the pigments showed a significant increase in viscosity (except Bohemian green earth). The increases in viscosity ranged from 20% to 40%, compared to the standard dolomite filler P60. Only Bohemian earth pigment had the same effect on viscosity as the standard filler. Increased viscosity is undesirable for packaging, application, sagging, and leveling. Reduced viscosity is equally undesirable, so modification with Bohemian earth provided the only acceptable outcome. On the other hand, the density of the paint was not significantly increased by the pigments (in the case of zinc-iron bronze and iron red), or as even slightly reduced (in the cases of Nicosia green and Bohemian green earth). This is a positive result, as it could diminish production costs-paints are sold by volumetric measure, whereas raw materials are sold by weigh measure. Figure 1 shows the effects of the studied pigments on the density and viscosity of the final products. Viscosity is a main factor in determining the application properties of paints. Waterborne paints are designed to have specific rheological properties, such as sagging resistance or good leveling on walls [40]. It is important to maintain viscosity while changing ingredients. As can be seen in Figure 1, all the pigments showed a significant increase in viscosity (except Bohemian green earth). The increases in viscosity ranged from 20% to 40%, compared to the standard dolomite filler P60. Only Bohemian earth pigment had the same effect on viscosity as the standard filler. Increased viscosity is undesirable for packaging, application, sagging, and leveling. Reduced viscosity is equally undesirable, so modification with Bohemian earth provided the only acceptable outcome. On the other hand, the density of the paint was not significantly increased by the pigments (in the case of zinc-iron bronze and iron red), or as even slightly reduced (in the cases of Nicosia green and Bohemian green earth). This is a positive result, as it could diminish production costs-paints are sold by volumetric measure, whereas raw materials are sold by weigh measure.

Gloss, Rub-out test and Substrate Adhesion
None of the chosen pigments affected gloss enough to change the character of the paint ( Table 1). As paint is formulated to match a specific type of gloss, new additives should not change the gloss of the product. The second part of table presents the adhesion class and percentage of the exfoliation of the studied pigments. It is important that pigments do not reduce adhesion to the substrate, as this can lead to peeling and the paint falling off. All of the studied pigments were placed in either the first-or second-best categories for substrate adhesion (exfoliation equal 0 means best, class 0, and exfoliation lesser than 5% means second-best class 1), meaning they did not affect the initial properties of the paint. As mentioned in the Methods section, the paint was only applied on cardboard. Although different substrates are known to yield different adhesion results [41], it was only meant to compare the same paint with no difference in binder, so use of one substrate was deemed to be sufficient.

Gloss, Rub-Out Test and Substrate Adhesion
None of the chosen pigments affected gloss enough to change the character of the paint ( Table 1). As paint is formulated to match a specific type of gloss, new additives should not change the gloss of the product. The second part of table presents the adhesion class and percentage of the exfoliation of the studied pigments. It is important that pigments do not reduce adhesion to the substrate, as this can lead to peeling and the paint falling off. All of the studied pigments were placed in either the first-or second-best categories for substrate adhesion (exfoliation equal 0 means best, class 0, and exfoliation lesser than 5% means second-best class 1), meaning they did not affect the initial properties of the paint. As mentioned in the Methods section, the paint was only applied on cardboard. Although different substrates are known to yield different adhesion results [41], it was only meant to compare the same paint with no difference in binder, so use of one substrate was deemed to be sufficient. No effect on the compatibility of the pigments was observed in the rub-out test. Each pigment was well incorporated in the paint, and no color differences were observed during application.

Scrub Test
As shown in Figure 2, the addition of the studied pigments did not have a major impact on the wet-scrub resistance of the paint. This means that pigment particles were well coated with the polymer and the surface of the coating remained unchanged relative to the standard formulation. It is not uncommon for slight changes in the paint formulation to affect wet-scrub resistance [42]. Therefore, this test should be conducted each time even minor modifications are made. If any of the tested pigments disturb the process of film formation, either chemically or mechanically, this will be observed as a decrease in wet-scrub resistance. All of the tested samples showed reduced thicknesses of between 7 and 10 µm, which is classified as class II scrub resistance following the PN-EN 13300 standard (for thickness loss not greater than 20 µm and not less than 5 µm). Therefore, the tested pigments did not interfere with the process of film formation, compared with the standard formulation. The obtained results of the scrub test seem be in agreement with the data obtained from Zetasizer apparatus (Appendix A Table A1). The size of the pigment particles was close to the size of the standard dolomite filler tested in this study. Particles bigger than standard filler could "stand out" and be removed more easily, causing bigger thickness loss, whereas particles sizes close to those of paint's fillers did not affect it significantly. No effect on the compatibility of the pigments was observed in the rub-out test. Each pigment was well incorporated in the paint, and no color differences were observed during application.

Scrub Test
As shown in Figure 2, the addition of the studied pigments did not have a major impact on the wet-scrub resistance of the paint. This means that pigment particles were well coated with the polymer and the surface of the coating remained unchanged relative to the standard formulation. It is not uncommon for slight changes in the paint formulation to affect wet-scrub resistance [42]. Therefore, this test should be conducted each time even minor modifications are made. If any of the tested pigments disturb the process of film formation, either chemically or mechanically, this will be observed as a decrease in wet-scrub resistance. All of the tested samples showed reduced thicknesses of between 7 and 10 µ m, which is classified as class II scrub resistance following the PN-EN 13300 standard (for thickness loss not greater than 20 µ m and not less than 5 µ m). Therefore, the tested pigments did not interfere with the process of film formation, compared with the standard formulation. The obtained results of the scrub test seem be in agreement with the data obtained from Zetasizer apparatus (Appendix A Table A1). The size of the pigment particles was close to the size of the standard dolomite filler tested in this study. Particles bigger than standard filler could "stand out" and be removed more easily, causing bigger thickness loss, whereas particles sizes close to those of paint's fillers did not affect it significantly.

Water Absorption
Water absorption was measured three times due to possibility of the emulsifier being rinsed out of sample, which can affect the test outcome. This explains why the second and third series differ quite significantly from first series. The results presented in Figure 3 show that most of the samples can be assigned to class II for water absorption. Bohemian green earth pigment improved the performance of the paint by reducing water uptake to the point of reaching class III water absorption. This improvement may be explained by small-sized pigment particles sealing some of the pores, as well as the presence of the silicon in the structure of the Bohemian green earth pigment. For outdoor applications, reduced water absorption is an important factor, as it restricts moisture transport to the inside of the wall, making it more durable and less vulnerable to cracks and deformations. Bohemian green earth reduced water absorption the most, placing it in highest possible class III according to the PN 76/C-81521 standard. This could have a positive effect on production costs by reducing the use of hydrophobic agents, which are usually expensive. the point of reaching class III water absorption. This improvement may be explained by small-sized pigment particles sealing some of the pores, as well as the presence of the silicon in the structure of the Bohemian green earth pigment. For outdoor applications, reduced water absorption is an important factor, as it restricts moisture transport to the inside of the wall, making it more durable and less vulnerable to cracks and deformations. Bohemian green earth reduced water absorption the most, placing it in highest possible class III according to the PN 76/C-81521 standard. This could have a positive effect on production costs by reducing the use of hydrophobic agents, which are usually expensive.  Figure 4 presents the effects of the studied pigments on the vapor permeability and open time of the paint. As can be seen, there was a slight reduction in the vapor permeability of paint filled with Bohemian green pigment. However, the assigned class of the paint was unchanged. It should be noted that vapor permeability is often associated with lower water uptake, so improving the class for water absorption without changing the class for the vapor permeability test can be considered a success. Keeping the same level of vapor permeability means the paint is not more likely to keep water vapor inside the walls, making the walls more vulnerable to degradation. Moreover, none of the pigments   Figure 4 presents the effects of the studied pigments on the vapor permeability and open time of the paint. As can be seen, there was a slight reduction in the vapor permeability of paint filled with Bohemian green pigment. However, the assigned class of the paint was unchanged. It should be noted that vapor permeability is often associated with lower water uptake, so improving the class for water absorption without changing the class for the vapor permeability test can be considered a success. Keeping the same level of vapor permeability means the paint is not more likely to keep water vapor inside the walls, making the walls more vulnerable to degradation. Moreover, none of the pigments had a

Contrast
Visual differences are observable by the naked eye when at least dE = 1.0. This means all of the pigments performed well in terms of mating properties. This outcome was expected as the paint contains titanium oxide white, which has an excellent mating capability. However, this result alone is not enough to ensure sufficient mating. All of the studied pigments presented satisfying mating capabilities when added to paint (Table 2). Importantly, the dry pigments performed even better than their commercial equivalents, as evidenced by lower contrast values.

Contrast
Visual differences are observable by the naked eye when at least dE = 1.0. This means all of the pigments performed well in terms of mating properties. This outcome was expected as the paint contains titanium oxide white, which has an excellent mating capability. However, this result alone is not enough to ensure sufficient mating. All of the studied pigments presented satisfying mating capabilities when added to paint (Table 2). Importantly, the dry pigments performed even better than their commercial equivalents, as evidenced by lower contrast values.

UV Aging
Paint samples containing the dry pigments were further exposed to long-term UV aging simulation tests. The color changes were monitored after 150 and 500 h of UV aging. The results are presented in Figure 5. Generally, the color changes (∆E) were too slight to be noticed by the naked eye even after 500 h of testing. This indicates excellent resistance to UV light. The most satisfactory result was observed for iron-red pigment, with ∆E of less than 2 even after 500 h of aging. This result is very similar to the commercial pigment RX10, and much better than the commercial red RH6. Typically, inorganic pigments exhibit better stability against light in paints compared to their organic equivalents. However, the Bohemian green earth performed worse than its commercial counterpart, and Nicosia green showed the worst performance overall, changing color twice as much as commercial green GX11. This is unfortunate, since maintaining color is an important trait for decorative coatings, and alternatives to expensive green pigments are highly desired. The spinelbronze pigment aged rapidly over the first 150 h. However, after reaching ∆E~4, it did not degrade much further. It is generally acceptable for pigments to lose their color properties under sunlight, although this degradation should not occur too quickly. In this case, the degradation of the samples containing dry pigments was comparable to the degradation of the samples containing commercial pigments, meaning that the dry pigment samples were acceptable in terms of UV stability.
Nicosia green showed the worst performance overall, changing color twice as much as commercial green GX11. This is unfortunate, since maintaining color is an important trait for decorative coatings, and alternatives to expensive green pigments are highly desired. The spinel-bronze pigment aged rapidly over the first 150 h. However, after reaching ΔE~4, it did not degrade much further. It is generally acceptable for pigments to lose their color properties under sunlight, although this degradation should not occur too quickly. In this case, the degradation of the samples containing dry pigments was comparable to the degradation of the samples containing commercial pigments, meaning that the dry pigment samples were acceptable in terms of UV stability.

Figure 5.
Color change (ΔE) of paint samples exposed to different durations of UV aging.

New Paint Formulation (Viscosity Measurements, Storage Stability, Wet-scrub Resistance).
Based on the results of our preliminary studies, Bohemian green earth was selected as the best performing and most promising coloring additive. The deciding factor was

New Paint Formulation (Viscosity Measurements, Storage Stability, Wet-Scrub Resistance)
Based on the results of our preliminary studies, Bohemian green earth was selected as the best performing and most promising coloring additive. The deciding factor was that it maintained the same level of viscosity as the original paint, so it did not change its application properties of the paint. Viscosity is the easiest change to observe and has the most influence on the basic properties of paint. Moreover, Bohemian green earth did not change and even improved some of the other characteristics of the paint and even improved some of them, including water absorption, making the pigment a functional additive.
After selecting the most satisfactory pigment, a new paint formulation was prepared containing 5% weight content of Bohemian green earth (see Table 3). The standard formulation was used as a reference (Table 3). Standard tests were performed on both samples. Viscosity was measured twice, immediately after sample preparation and after 24 h. Additionally, storage stability and wet-scrub resistance tests were performed. A viscosity curve was prepared using an Anton-Paar MCR102 Rheometer.
The viscosity of the newly-formulated paint was measured according to the same method used in our preliminary studies, using a Brookfield viscometer. Measurements were taken immediately after preparation of the paint and again after 24 h to determine if any loss of viscosity had occurred. The results are shown in Table 4. As can be seen, the impact on viscosity is negligible. As our previous findings demonstrated, the pigment does not have any major effect on the basic rheological properties of the original formulation. The slight increase in viscosity over 24 h implies thixotropic behavior, which was maintained after modification.
Viscosity was measured after placing the samples in an incubator for 2 weeks at a temperature of 50 • C. The samples were observed to detect the occurrence of any sedimentation of the dispersed phase. The aim was to simulate the storage behavior of the final product. The results are presented in Table 4.
Wet-scrub resistance was tested according to the same method used in our preliminary research. The results are presented in Table 5. Samples were applied to a 300-µm substrate and left for 28 days at room temperature to dry out. The thickness of each sample was measured before and after scrubbing. Each sample was subjected to 200 cycles of scrubbing using sponges and 0.5% ABSNa solution. Scrub resistance was evaluated according to PN-EN 13300 classifications. As can be seen in Table 5, modification with Bohemian green earth did not affect the scrub resistance of the original formulation.

Rheological Profile of the Newly-Formulated Paint
In the last stage of the research, the rheological properties of the newly-formulated paint were evaluated using an Anton-Paar MCR102 (Graz, Austria) rotational rheometer with a plate-plate measuring system (two 25 mm diameter plates rotating in parallel with the sample placed between them). The samples were subjected to the rotational movement of the upper plate with an increasing shear rate. The rheological results were recorded on a viscosity-shear rate graph ( Figure 6). The viscosity-shear rate graph can be divided into three areas. Shear rates of 0.01-1 s −1 correspond to storage stability. Shear rates of 1-10 s −1 correspond to in-can mixing and leveling. Higher shear rates correspond to brush loading and application [40]. In the last stage of the research, the rheological properties of the newly-formulated paint were evaluated using an Anton-Paar MCR102 (Graz, Austria) rotational rheometer with a plate-plate measuring system (two 25 mm diameter plates rotating in parallel with the sample placed between them). The samples were subjected to the rotational movement of the upper plate with an increasing shear rate. The rheological results were recorded on a viscosity-shear rate graph ( Figure 6). The viscosity-shear rate graph can be divided into three areas. Shear rates of 0.01-1 s −1 correspond to storage stability. Shear rates of 1-10 s −1 correspond to in-can mixing and leveling. Higher shear rates correspond to brush loading and application [40].  As can be seen in Figure 6, the modified paint presents higher initial viscosity compared to the standard formulation, although it liquefies more easily at low shear-rate values (0.01-1), suggesting it might present less viscous behavior during storage. At mid-shear rates (1-10), the paints show similar viscosity, meaning they would present similar behavior while mixing in the can. However, the steeper slope in the case of the modified paint might indicate a higher tendency for sagging. Finally, in the application range (10-100) the viscosity of the samples is indistinguishable as the graphs are identical.

Conclusions
This work investigated the possible use of dry pigments as replacements for currently used colorants, even without first being liquified into pigment pastes. The best results were obtained with the addition of Bohemian green earth pigment, which had no major impact on the rheological characteristics of the original paint, meaning it can be used without being made into a pigment paste. This is a huge advantage, as green pigments tend to be the most expensive additives. Nonetheless, any change in the formulas of commercial paints, especially related to the color, brings additional costs. Each new addition to the formula should be subjected to tests to determine its impact on important properties, and its possible benefits for paint quality.  Acknowledgments: This work has been completed while the first author was the Doctoral Candidate in the Interdisciplinary Doctoral School at the Lodz University of Technology, Poland.

Conflicts of Interest:
The authors declare no conflict of interest.


text:
THE PRODUCTION OF TRANSGENIC TOBACCO PLANTS OVEREXPRESSING OAK DEHYDRIN GENE

focused on the production of transgenic tobacco plants

INTRODUCTION
Dehydrins (PF00257) belong to a group of LEA proteins (Jaspard et al., 2012). They are thermostable and highly hydrophilic proteins that are associated mainly with later stages of plant embryogenesis (Amara et al., 2014). Besides that, many studies indicate a role of dehydrins in plant stress tolerance. They are accumulated under low temperature, water deficiency, osmotic or heavy metal stress (Close, 1997 Dehydrins are defined by the presence of a lysine-rich motif called the K-segment (EKKGIMDKIKEKLPG). The K-segment can form an amphiphilic α-helix that may contribute to the stabilisation of the proteins and cellular membranes. Some dehydrins contain also motifs called Y-and S-segments. The Y-segment could be a likely place for binding nucleotides. The S-segment consists of serine residues and acts as a site of phosphorylation. Based on the presence of these conserved segments, dehydrins are classified into five main subclasses: Kn, SKn, KnS, YnKn and YnSKn (Battaglia et al., 2008). In general, dehydrins are considered multifunctional proteins, but not all dehydrins perform all functions. Some functions are common to many dehydrins, while some may be specific, e.g. ability to bind nucleic acids (Boddington et al., 2019). They can act as cryoprotectants, chaperones, or ROS scavengers. Dehydrins can bind free metal ions, phospholipids, or proteins (Hara, 2010;Eriksson et al., 2016;Hara et al., 2016). The roles of dehydrins in plant development and stress tolerance have been studied either in their native organism (Eriksson et al., 2016;Hara et al., 2016) or using a system of transgenic plants (Xu et al., 2008;Liu et al., 2019). Despite intensive research on dehydrin genes over the past 20 years, dehydrins in woody plants are relatively little explored. Trees located in the temperate zone need to survive the cold season, so they must tolerate the shortening of daylight and low temperatures (Welling et al., 2004). Thus, dehydrins in woody plants are mainly associated with freezing tolerance and dormancy. In the genus Quercus, dehydrins have been studied in the species Q. robur, Q. petraea, Q. ilex, or Q. cerris. In the species Q. robur, three dehydrins QrDhn1 (YSK3-type), QrDhn2 (K4-type), and QrDhn3 (K3-type) have been identified so far (Šunderlíková et al., 2009). Northern blot analysis revealed, that these dehydrins respond to altered water and osmotic conditions. In this work, we prepared a plant transformation vector pMK and used it for Agrobacterium-mediated genetic modification of tobacco. The plant transformation vector pMK contained in its T-DNA an expression cassette consisting of the oak dehydrin gene Dhn3 under the control of a constitutive double dCaMV 35S promoter. The sequence of the oak dehydrin gene (AY607707.1) was isolated by PCR approach. Transgenic plants overexpressing the oak dehydrin were regenerated under the selection pressure of kanamycin. The transgenic nature of regenerated plants was confirmed by molecular analyses.

Plant transformation vector preparation
The sequence of the oak dehydrin gene (AY607707.1) was isolated by PCR approach using the primer set P1/P2 (Table 1). The genomic DNA was extracted from the leaves of Quercus robur according to the protocol by Chen et al. (1992). The PCR reaction mixture (25 µl) consisted of 100-200 ng DNA, 200 μM dNTPs, 20 pmol of the primers, 1× PCR buffer and 1 unit of FIREPol® DNA polymerase (Solis BioDyne). The first PCR step of 95˚C for 4 minutes was followed by 35 cycles: 95˚C 45 s; 64˚C 45 s and 72˚C for 2 minutes. The last step was performed at 72˚C for 10 minutes. The PCR product was isolated from the gel using QIAquick Gel Extraction Kit (Qiagen) and commercially sequenced. The PCR product was ligated into the cloning vector pJET1.2 (CloneJet PCR Cloning Kit, Thermo Fisher Scientific). Then, the sequence Dhn3 as a 350 bp XhoI-XbaI fragment from the plasmid pJET1.2-Dhn3 and the polyA sequence as a 268 bp XbaI-HindIII fragment from the plasmid pRT100 (Töpfer et al.;1987) were ligated into the cloning vector pSK+. The plant transformation vector pMK ( Figure 1A) was prepared by the ligation of the Dhn3/polyA sequence as a 648 bp EcoRI-NcoI fragment and the sequence dCaMV35S as a 736 bp NcoI-HindIII fragment into the EcoRI a HindIII digested plasmid pBinPlus (Van Engelen, 1995). The plasmid pMK was transformed into Agrobacterium tumefaciens LBA 4404 and used in the transformation experiments.

Tobacco transformation and regeneration
Tobacco plants (Nicotiana tabacum L. cv. Petit Havana SR1) were cultivated in in vitro conditions at 20 ± 2 o C with a day length of 16 h under 50 µE m -2 s -1 light intensity. Leaf segments of six-weeks-old in vitro tobacco plants were transformed via Agrobacterium-mediated transformation as was described by Polóniová et al. (2015). Transformed tobacco cells were regenerated under the selection pressure of kanamycin (50 mg/L). The transformation efficiency was evaluated as the number of shoots that were rooted in the presence of kanamycin with respect to the total number of explants used in the experiment. This work is focused on the production of transgenic tobacco plants overexpressing the oak dehydrin gene AY607707.1. Transgenic tobacco plants were generated via Agrobacterium-mediated transformation. The T-DNA of the plant transformation vector pMK contained the dehydrin gene fused to the constitutive double dCaMV35S promoter and the selectable neomycin phosphotransferase gene. The sequence of the dehydrin gene was isolated from Quercus robur by PCR approach and cloned. The constructed binary vector pMK was introduced into Agrobacterium tumefaciens LBA4404 and used in the transformation experiments. Transgenic plants were generated with an efficiency of 32.7%. PCR analyses confirmed the transgenic nature of regenerated T0 plants. The expression of the oak dehydrin gene was proved by RT-PCR and quantified by qPCR analyses.

Molecular characterisation of transgenic plants
DNA was isolated from the leaves of tobacco plants using a DNeasy® Plant Mini Kit (QIAGEN). PCR analyses were performed with the primers P1/P2, P3/P4, and P5/P6 that were designed to confirm the presence of the dehydrin Dhn3 and the selectable nptII genes; and the actin as a housekeeping gene, respectively (Table  1). The PCR mixture and PCR reaction conditions are given above. Total RNA was isolated from leaves of tobacco plants using RNeasy® Plant Mini Kit (QIAGEN). RNA was reverse transcribed using Maxima First Strand cDNA Synthesis Kit (ThermoFisher Scientific). RT-PCR analyses were realised with the primer sets P7/P8 and P5/P6 to confirm the presence of genes Dhn3 and actin, respectively (Table 1). The PCR reaction mixture (25 µl) consisted of 20 ng cDNA, 200 μM dNTPs, 20 pmol of the primers, 1× PCR buffer and 1 unit of FIREPol® DNA polymerase (Solis BioDyne). The PCR reaction conditions are given above. The qPCR analyses were realised with the primer sets P9/P10 and P5/P6 that amplified fragments corresponding to the genes Dhn3 and actin, respectively ( Table 1). The analyses were performed using a LightCycler® Nano System (ROCHE). The qPCR reaction conditions were according to the manufacturer´s instructions (Luminaris HiGreen qPCR Master Mix, ThermoFisher Scientific). The expression of the dehydrin gene Dhn3 was quantified using the ΔCt method.

RESULTS AND DISCUSSION
Dehydrins belong to a group of hydrophilic proteins that are associated with the stress response in plants. Their main role is thought to be to prevent protein inactivation and aggregation. Dehydrins are characterised by the presence of conservative regions called K-, S-and Y-segments. Based on their structure, they can be divided into five types YnSKn, YnKn, SKn, Kn and KnS, which to some extent determine their function (Battaglia et al., 2008). In this study, we isolated genomic DNA from the leaves of Quercus robur and used it as a template for PCR to amplify the sequence of the oak dehydrin gene (Dhn3) ( Figure 1C). The isolated intronless sequence Dhn3 was of a size 346 bp. The deduced Dhn3 protein contains 112 amino-acid residues with a molecular weight of about 12.3 kDa and a predicted isoelectric point of 9.66. The dehydrin Dhn3 contains three repeats of K segments and structurally belongs to the Kn-type of dehydrins. The plant transformation vector pMK ( Figure 1A) was created by the introduction of the expression cassette consisting of the Dhn3 gene fused to the constitutive dCaMV 35S promoter as a 736 bp dCaMV 35S/Dhn3/polyA fragment into the binary vector pBinPlus. The plasmid pBinPlus is a derivate of the binary vector pBin19 (Bevan et al., 1984). The T-DNA of the plasmid pBinPlus contains a selectable marker nptII gene that is placed at the left T-DNA border (LB). Given that the transfer of T-DNA is polar (Lee and Gelvin, 2008), the location of the nptII gene at the LB border increases the likelihood of regenerating transgenic plants with an intact T-DNA. The identity of the plasmid pMK was confirmed by restriction analyses (Figure 1B, Figure 1D). Transgenic tobacco plants were generated via Agrobacterium-mediated transformation. Leaf explants (Figure 2A) were infected with bacterial inoculum A. tumefaciens LBA4404/pMK. Since the pMK-T-DNA contains the nptII marker gene, which encodes resistance to antibiotics such as kanamycin or neomycin, transformed cells were regenerated under the selection pressure of kanamycin (50 mg/L). We used a total of 104 explants for transformation experiments. The first calli appeared after two weeks ( Figure 2B). The first shoots began to form 4-6 weeks after transformation ( Putative transgenic T0 plants ( Figure 2D) were subjected to molecular analyses to confirm their transgenic nature (Figure 3). The presence of the dehydrin Dhn3 and the marker nptII genes were confirmed with the primer sets P1/P2 and P3/P4 that were designed to amplify the fragments of the sizes 346 bp and 552 bp, respectively ( Figure 3A). The fragments of the expected sizes were identified in all (12) analysed T0 plants. We did not observe the PCR products in wild-type tobacco plants. RT-PCR analyses were used to detect the expression of the dehydrin Dhn3 gene in transgenic T0 plants. Analyses were performed with the primers P7/P8 that were designed to amplify a 251 bp fragment ( Figure 3B). The PCR product of the expected size was detected in all (12) analysed T0 plants. The relative expression levels of the dehydrin Dhn3 in transgenic tobacco were quantified by qPCR with the primer set P9/P10. As shown in Figure 3C, the relative expression level of the Dhn3 was variable depending on the transgenic T0 line.

CONCLUSION
Transgenic tobacco plants overexpressing oak dehydrin gene were generated via Agrobacterium-mediated transformation. The T-DNA of the plant transformation vector pMK contained the oak dehydrin gene under the control of the constitutive dCaMV 35S promoter and the selectable marker nptII gene. The sequence of the oak dehydrin gene Dhn3 from the Quercus robur was isolated based on the PCR approach and cloned. The constructed plasmid pMK was transformed into A. tumefaciens LBA 4404 and used in the transformation experiments. Transgenic plants overexpressing the dehydrin gene Dhn3 were generated with an efficiency of 32.7%. Transgenic nature of regenerated T0 plants was confirmed by PCR analyses. The expression of the Dhn3 gene was proved by RT-PCR and quantified by qPCR analyses. Transgenic plants with the highest expression levels of the transgene will be used in further experiments focused on the effect of overexpression of the oak dehydrin gene on plant stress tolerance.