The effect of gelatin, used as a biostimulant, was investigated on plant growth in greenhouse studies. Biostimulants are materials that stimulate plant growth, and gelatin, an animal protein hydrolysate, is classified as one type of biostimulant. Gelatin has a unique amino acid composition with a high percentage of proline and hydroxyproline. In a series of experiments gelatin capsules (#3 hard gelatin) containing 7.1 mg nitrogen each, were placed adjacent to seeds of different crop species, at sowing time in individual growing containers and several growth parameters were measured. Different types of hydrolyzed collagen, including granulated gelatin, gelatin hydrolysate, and amino acid mixtures simulating the composition of gelatin were compared on cucumber plant growth. In addition, amino acid mixtures without proline, hydroxyproline, or applied in combination were investigated on cucumber growth. All capsule treatments significantly enhanced crop growth compared to the non-treated control. The treatment with two gelatin capsules placed adjacent to each seed increased shoot dry weight of cucumber, pepper, broccoli, tomato, arugula, and field corn, by 138, 244, 50, 45, 41, and 18 percent, respectively. In an experiment with cucumber alone, there was a positive linear relationship between the number of gelatin capsules from 0 to 3 capsules on plant growth and plant nitrogen content. Cucumber growth and plant nitrogen content was greater from the hydrolyzed collagen treatment compared with the low molecular weight gelatin hydrolysate, a mixture of amino acids or urea and all treatments provided an equivalent amount of nitrogen. Proline and/or hydroxyproline were not responsible for the biostimulant effect. In summary, gelatin provided nitrogen that enhanced plant growth. Moreover, gelatin was an effective biostimulant as the plant growth and nitrogen content was greater from two gelatin capsules compared to amino acid mixture of the same proportion and amount as the gelatin. Seed enhancement is a term widely used in the industry to describe beneficial techniques performed to seeds post-harvest, but prior to sowing . Seed enTable 1) and the molecular size distribution for amino acids present in hydrolyzed gelatin ranges from 89.1 to 204.2 g mol−1 (Table 1). A gelatin molecule is composed of an average of 1,000 amino acids per chain and there are a few disulfide bonds in gelatin molecular structure , an animal membrane hydrolysate, showed membrane stability and increased photosynthetic capacity when subjected to high temperature stress . Seeds were sown in 10 cm × 10 cm × 12 cm  plastic pots filled with Cornell Peat-Lite Mix A medium routinely used for seedlings or bedding plants. Peat-lite mix A is a combination of 1:2:2 (vol/vol) perlite: sphagnum peat moss: horticultural grade vermiculite with 4 kg of dolomitic limestone, 0.33 kg of nitrogen (N) and potassium (K2O), and 0.16 kg of phosphate (P2O5) per cubic meter of mix , arugula “Astro” , broccoli “Centura” , tomato “Talladega” , pepper “Boynton Bell” , and field corn “Cornell D2901”  were grown in a controlled greenhouse maintained at 24°C/21°C temperature with 14/10 h photoperiod at New York State Agricultural Experiment Station in Geneva, NY, United States. Five gelatin capsule treatments  were evaluated using #3 hard-gel animal based gelatin capsules , referred throughout paper as a gelatin capsule or gelatin capsule treatments. One gelatin capsule contains 7.1 mg nitrogen that is equivalent to 40 mg protein . Capsuler of mix . Five reFigure 1). Five replications of each treatment were placed in a random block design in the greenhouse, with 15 samples per replication. The plants were harvested 28 days after emergence and seven physiological parameters were measured . Total leaf area was measured using CI-202 Leaf Area Meter .Cucumber seeds “Vlaspik”  were grown in a controlled greenhouse maintained at 24°C/21°C temperature with 14/10 h photoperiod at New York State Agricultural Experiment Station in Geneva, NY, United States. Four gelatin capsule treatments  were evaluated using #3 hard-gel gelatin capsules . Capsules were placed adjacent to each seed in a 10 cm square pot: control (no capsule), half capsule (0.5), whole capsule (1.0), two capsules (2.0). The placement of two capsules is illustrated in relation to a cucumber seed  to a particle size of 2 mm. A 100 mg sample was sent to Cornell University Stable Isotope Laboratory  for elemental analysis to determine total nitrogen amount in plant tissue.Table 2).Ten gram samples of three different hydrolyzed collagen types: granulated 220 bloom gelatin, granulated gelatin capsule, and gelatin hydrolysate were submitted to Eastman Gelatine Co.  for MWD analysis. MWD of hydrolyzed collagen was performed by high-performance liquid chromatography (HPLC) in the aqueous size exclusion mode (AEC). Gelatin samples were dissolved in the chromatographic eluent along with a phosphate buffer containing sodium dodecyl sulfate (SDS). Molecular weight fractions from experimental samples were separated on a TosoHaas TSK-Gel size exclusion column . The effluent was monitored with a UV detector set at 220 nm. Known molecular weight standards were run to prepare a calibration curve, which was constructed by plotting the log of these molecular weights versus the retention time. The MWDs of unknown gelatin samples were then determined from the linear portion of the calibration curve. Results were reported in terms of relative area percent of the five molecular weight regions (Table 1). Bloom 220 gelatin was chosen as it has the same bloom value as the gelatin capsule. Gelatin hydrolysate was chosen for its small molecular size and its unique characteristic of lack of gelling capability. Two gelatin capsules were placed adjacent to each seed in a 10 cm × 10 cm × 12 cm pot . The hydrolyzed bloom 220 gelatin, gelatin hydrolysate, urea and amino acid mixture were weighed to the equivalent weight of two gelatin capsules (90 mg) and applied to the seed as powders in the pot. Each treatment had a total of 75 experimental units arranged in five replications of 15 experimental units per block, and the treatments were placed in a random block design on the greenhouse bench. The plants were harvested 28 days after emergence and leaf area and dry weight were measured. The plant nitrogen content was determined as previously described. The contribution of the gelatin treatments to nitrogen applied per plant was determined by capsule weight with 14% moisture content , gelatin hydrolysate , urea , and an amino acid mixture that simulated the same composition as gelatin  . To detect the statistical significance of differences between means, the Tukey’s HSD test at a significance level of α = 0.05 was performed. Also for experiment 1, capsule treatments were grouped by crop species for analysis. Linear regression was used to study cucumber plant total nitrogen response to increasing nitrogen inputs from gelatin capsules (Experiment 2.2). For all experiments raw data were used for analyses. The statistical analysis was performed using the statistical program JMP Pro 11 .2 or g, respectively, and percent change with respect to the control. All of the crops exhibited significant increases in total leaf area with the gelatin capsule treatment. The magnitude of the effect of the capsule treatment on the total leaf area was specific to each crop . Cucumber had the most significant increases in total leaf area (p < 0.0001) with 25 cm2 (39% increase) with half capsule treatment, 64 cm2 (102% increase) with one capsule treatment, and 121 cm2 (193% increase) with two capsule treatment compared to the control. The maximum total leaf area in cucumber was achieved with the three capsule treatment with 223 cm2 (356% increase) compared to the control. Arugula and broccoli exhibited a similar increase in total leaf area with capsule treatments compared to cucumber. Tomato showed a significant increase in total leaf area with the addition of capsule treatments (p < 0.0001). For tomato there was a 50 cm2 (123% increase) with one capsule, and maximum increase of 52 cm2 (126%) with two capsule treatment, and a slight decrease in total leaf area with the three capsule treatment with 48 cm2 (116% increase). Pepper plants exhibited a similar trend as tomato. Corn did not respond as dramatically as the other crops and an increase of 12 cm2 (20%) was observed with the two capsule treatment.Enhanced plant growth was observed in the above ground portions of plants in treatments with gelatin capsules placed adjacent to seeds at the time of sowing. Results for total leaf area, and dry weight were reported as cmp < 0.005)  compared to the non-treated control. Cucumber had the most significant increases in dry weight (p < 0.0001) with 0.1 g (30% increase) with half capsule treatment, 0.29 g (84% increase) with one capsule treatment, and 0.47 g (138% increase) with the two capsule treatment compared to the control. The maximum dry weight in cucumber was achieved with the three capsule treatment with 0.86 g (251% increase) compared to the control. Arugula and broccoli exhibited similar increases in dry weight with capsule treatments compared to cucumber. Tomato had significant increase in dry weight with the addition of capsule treatments (p < 0.0001). There was 0.14 g (56%) maximum increase with one capsule, followed by a slight decrease in dry weight with the two and three capsule treatments with 0.11 g (45%) and 0.10 g (37%) increase, respectively. Pepper had a similar trend as tomato.Similar to total leaf area, all of the crops exhibited significant increases in dry weight with the capsule treatments . The effects of the capsule treatments were not consistent for all plant parts. The above ground parts of the plant exhibited a significant increase (p < 0.05) with the addition of two capsule treatments. Petiole length increased 37%, total leaf area had a 48% increase, shoot fresh weight had a 45% increase . Below ground biomass did not reflect the same growth increase as measured for the above ground parts of the plant. There was no significant difference in growth for the below ground part of the cucumber plant.The effect of capsule treatments on cucumber plant was investigated by measuring seven different growth parameters  28 days after emergence. The half capsule treatment had the least effect on plant growth, and the two capsule treatments had the greatest effect on the overall cucumber plant growth compared to the non-treated control. An additive effect of the hard-gel capsule treatment was measured in plant height, petiole length, total leaf area, fresh and dry shoot weight. Data are presented as percent difference of the seven growth parameters compared to the control (p < 0.0001) . The three capsule treatment exhibited the greatest percent increase in total nitrogen amount with 21.7 mg (617% increase), followed by treatment with two capsules 11.3 mg (321% increase), one capsule with 6.3 mg (178% increase), and the half capsule treatment with 1.8 mg (51%). Nitrogen amount recovered from plant tissues was significantly related to the nitrogen supplied by the gelatin treatments (R2 = 97.0%) .Cucumber plants treated with gelatin capsules exhibited an increase in total nitrogen amount (mg) in plant tissue compared to the control . The gelatin capsule had an elution profile similar to bloom 220 gelatin. The gelatin capsule and bloom 220 gelatin, termed granulated hydrolyzed collagen both had high molecular weight (HMW), molecular weight region (MWR) above 250,000 g mol−1, of 25 and 20%, respectively. The beta fraction, MWR between 250,000 and 150,000 g mol−1, was 16% for gelatin capsule, and 14% for granulated hydrolyzed collagen. The alpha fraction, MWR between 150,000 and 50,000 g mol−1, was 35% for gelatin capsule, and 33% for granulated hydrolyzed collagen. The sub alpha fraction, MWR between 50,000 and 20,000 g mol−1, was 16% for gelatin capsule, and 21.1% for the granulated hydrolyzed collagen. The low molecular weight fraction, MWR < 20,000 g mol−1, was 7.5% for gelatin capsule, and 11% for granulated hydrolyzed collagen. There were significant differences (p < 0.001) in the molecular distribution of the three hydrolyzed collagens. No significant difference was detected between gelatin capsule and granulated hydrolyzed collagen, while there were significant differences (p < 0.001) between gelatin capsule, granulated hydrolyzed collagen and gelatin hydrolysate.According to the size exclusion elution profile of the different hydrolyzed collagens, gelatin hydrolysate had the smallest molecular weight, and 100% of its fraction was in the low molecular weight fraction . The urea and amino acid mixture had similar dry weight, which was significantly greater than the non-treated control. The bloom 220 had the highest N amount per plant sample at 7.7 mg (140% increase), which was not significantly different than the two gelatin capsule treatment . There were no differences in the amount of nitrogen between the urea, gelatin hydrolysate and amino acid mixture treatments.The effects of four types of hydrolyzed collagen on plant growth were investigated by measuring total leaf area and dry weight. Only dry weight data are presented. The two capsules and bloom 220 treatments had the greatest dry weight compared to other treatments . Therefore, proline and hydroxyproline were not the primary amino acids responsible for the plant growth promotion from gelatin capsule treatments.Proline and hydroxyproline were tested separately or in combination as a biostimulant. Total leaf area and dry weight were not different for the amino acid mixture containing proline, hydroxyproline or their combination, in comparison with the amino acid mixture without proline and/or hydroxyproline . Two gelatin capsules placed adjacent to each seed increased dry weight of cucumber, pepper, broccoli, tomato, arugula, and field corn, by 138, 244, 50, 45, 41, and 18 percent, respectively, compared to the non-treated control . Therefore, all crops investigated exhibited enhanced plant growth; however, the growth enhancement, measured by the percent increase in dry weight, compared to the non-treated control differed by species. This may be attributed to the uptake mechanism of hydrolyzed collagen that is the main ingredient in the gelatin capsules. Hydrolyzed collagen, a polypeptide, is comprised of amino acids, and the amino acids can be used as a nitrogen source in plant nutrition  exhibited increased total leaf area and dry weight in response to gelatin capsule treatments (utrition . Plants utrition .Sorghum bicolor), rice (Oryza sativa), maize (Zea mays), and pearl millet (Pennisetum glaucum). In their study, seedlings were grown without N, or with 500 mg N kg−1 applied to soil as ammonium nitrate, rice bran or a mixture of rice bran and straw. Shoot growth and N uptake in maize and pearl millet were correlated with the inorganic N (ammonium and nitrate) concentration in the soil, suggesting that these species depend on, or have a preference for, inorganic N. In contrast, shoot growth and N uptake patterns in sorghum and rice indicated that these two species could compensate for low inorganic N levels by taking up organic nitrogen (proteins) from the rice bran and straw mixtures. Analysis of N uptake in solution culture experiments confirmed that sorghum and rice roots were more able to absorb N in the form of protein than maize and pearl millet (Fragaria fragaria) and wild strawberry (F. chiloensis and F. virginiana). Exogenous glycine-N, NH4+-N, and NO3−-N was supplied to the three species of strawberry. The domestic strawberry only took up a small portion of N from the amino acids, while the wild species took up significantly more glycine-N and NH4+-N. These results show that utilization of organic N in the form of proteins and amino acids can be species specific.Previous reports have shown that the uptake of amino acids and proteins can vary between plant species . Okamotol millet . Reeve eFigure 4). Different doses of gelatin capsules had no significant effect on root growth compared to non-treated controls . The lack of the biostimulant effect on root growth may be related to the source of the protein in gelatin or the concentration and type that was available in the growing medium.In the study reported here, cucumber exhibited the largest growth response from gelatin capsule treatments than the other crops evaluated in the study. However, the gelatin capsule treatments only increased plant growth in the above ground biomass: plant height, petiole length, total leaf area, and shoot dry weight (Cucumis melo L.). In their experiments, five rates  of a biopolymer biostimulant were applied as a substrate drench. Total root dry matter, maximum root length, and root surface area in biostimulant treated plants were the greatest at 0.24 mL plant−1, but decreased at 0.48 mL plant−1. Arabidopsis and found that addition of low (1.6–5 μM) to intermediate (15–23 μM) concentrations of bovine serum albumen (BSA) protein increased root dry weight, root length and thickness, and root hair length. However, root growth was inhibited at the high concentration of BSA (45 μM).Research has shown that both the protein source and concentration applied can influence root growth and development. Figure 5). Thus the increase in nitrogen in the plant tissue was partially attributed to increased nitrogen supplied by the gelatin capsule treatments. However, the results suggest that growth promotion induced by the gelatin capsules was not caused by the N fertility alone as the treatment with two gelatin capsules had greater dry weight than the amino acid mix with the same proportion and amount of amino acids as the gelatin treatment . To determine if the growth promotion by the gelatin capsules was caused by the fertilizer effect from the amino acids in the gelatin, urea was used as a treatment with the equivalent amount of N found in two gelatin capsules. Although urea has the potential to be phytotoxic, no adverse effect in these experiments measured as the growth response and nitrogen per plant were the same from the urea treatment and the amino acid mixture . Brassica oleracea L.) seeds as a seed coating resulted in an unexpected increase in N uptake. Application of soy flour (a plant-based biostimulant source of nitrogen) as a component of a seed-coating blend increased N uptake efficiency. Nitrogen, from the soy flour applied in the seed coatings ranged from 0.024 to 0.073 mg per seed, while the enhanced nitrogen per plant ranged from 1.7 to 8.5 mg. Nitrogen applied in the seed coating only accounted for 1–2% of the increased nitrogen in plants, indicating the soy flour acted as a biostimulant and not as a fertilizer.A linear relationship between the nitrogen supplied by the gelatin capsules and the nitrogen in cucumber tissue was observed (Table 2), and their distribution differs between gelatin types . Gelatin hydrolysate had the smallest MWD with 100% of the elution in the low molecular weight fraction of <20 kDa . Gelatin capsules used in these experiments had an elution profile similar to granulated hydrolyzed collagen, which suggests that they are structurally similar. This speculation is supported by the plant dry weight results from the different hydrolyzed collagens that were evaluated. There were no significant differences in dry weight of cucumber between application of two gelatin capsules, and granulated hydrolyzed collagen (Bloom 220), while the dry weight for gelatin hydrolysate was significantly less than the Bloom 220 treatment . One explanation for the difference in plant dry weight between the treatments may be the water solubility of the different hydrolyzed collagens. Both gelatin hydrolysate and the amino acid mix have a high water solubility. As the plants were watered in the greenhouse during the experiments, the treatments that were applied in dry powder form may have dissolved in the water and leached from the containers. The lower solubility collagens may have remained in the container for a longer period of time and thus available for plant growth resulting in a higher plant biomass. Additional research is warranted to investigate water solubility and duration of availability of the different treatments for plant uptake.The hydrolyzed collagens used in these experiments were grouped by molecular weight (Table 1). Figure 8). Therefore, proline and hydroxyproline were not the primary amino acids responsible for the plant growth promotion from gelatin capsule treatments.The underlying mechanism of plant growth enhancement in response to gelatin capsules remains unclear. Gelatin has a unique composition of amino acids, and is rich in proline and hydroxyproline (Protein hydrolysates have been shown to stimulate carbon and nitrogen metabolism and increase nitrogen assimilation in plants . NitrateHW performed the experiments and wrote many parts of the article. MA involved in data analysis, results interpretation, and writing the article. AT defined the scientific hypothesis and set up the experiments protocol and contributed to refinement and development of this paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Distichlis spicate and Pluchea absinthioides from the AD, and Gaultheria mucronata and Hieracium pilosella from PAT. The abundance and composition of their endophytic bacterial communities was determined by quantitative PCR and high–throughput sequencing of 16S rRNA, respectively. Results indicated that there was a greater abundance of 16S rRNA genes in plants from PAT (1013 to 1014 copies g−1 DNA), compared with those from AD (1010 to 1012 copies g−1 DNA). In the AD, a greater bacterial diversity, as estimated by Shannon index, was found in P. absinthioides, compared with D. spicata. In both ecosystems, the greater relative abundances of endophytes were mainly attributed to members of the phyla Proteobacteria (14% to 68%), Firmicutes (26% to 41%), Actinobacteria (6 to 23%) and Bacteroidetes (1% to 21%). Our observations revealed that most of operational taxonomic units (OTUs) were not shared between tissue samples of different plant species in both locations, suggesting the effect of the plant genotype (species) on the bacterial endophyte communities in Chilean extreme environments, where Bacillaceae and Enterobacteriacea could serve as keystone taxa as revealed our linear discriminant analysis.Several studies have demonstrated the relevance of endophytic bacteria on the growth and fitness of agriculturally-relevant plants. To our knowledge, however, little information is available on the composition, diversity, and interaction of endophytic bacterial communities in plants struggling for existence in the extreme environments of Chile, such as the Atacama Desert (AD) and Patagonia (PAT). The main objective of the present study was to analyze and compare the composition of endophytic bacterial communities associated with roots and leaves of representative plants growing in Chilean extreme environments. The plants sampled were:  Beneficial functions attributed to endophytic bacteria include plant growth promotion by supplying nutrients , protection against biotic-  and abiotic-stresses , detoxification of harmful compounds , and the production of bioactive compounds 4. Various endophytic microorganisms have been categorized as plant growth–promoting bacteria (PGPB) and they are currently used in the formulation of diverse bioproducts  or to modify and/or introduce beneficial bacteria into the plant phytomicrobiome for agricultural purposes6. To date, however, many microbiome studies have been done using model plant , commercially relevant plants for agriculture  and wild plant species  grown under laboratory, greenhouse and fields conditions8. Consequently, we only have limited knowledge on the composition and interactions of microbiota and plants, especially endophytic bacterial communities, on native plant vegetation growing in extreme environments, such as hot and/or cold deserts. Thus, our understanding on microbial interactions in plant holobiont will be key in the develop of efficient strategies for native plant conservation and/or exploit the full yield potential of crop plants under climate change scenario9.Numerous studies have revealed that bacteria living within plant tissues, collectively called endophytic bacteria, play a crucial role in the growth and fitness of a wide variety of monocot and dicot plant species, among othersEnterobacteriales, Actinomycetales, and Rhizobiales comprise dominant groups of the bacterial communities in the rhizosphere (the soil influenced by plant roots) of shrubs grown in AD and Patagonia (PAT), namely Atriplex sp. and Chuquiraga sp., respectively10. Results of this study also suggested that some isolates, belonging to the genera Enterobacteria, Pseudomonas, and Bacillus, were putative PGPB. The ability of the native isolates from AD to act as PGPB was confirmed by formulation and inoculation of a bacterial consortium onto plants. These studies revealed that wheat plants inoculated with the consortium produced greater biomass under water shortage and field conditions, compared with uninoculated seedlings11. A recent study also showed a greater protection against salt stress in wheat plants inoculated with rhizosphere bacteria isolated from Andean Altiplano native plant (Parastrephia quadrangularis) in AD12. However, these studies did not take into account the composition and interaction of native endophytic bacteria in Chilean extreme environments, as well as their potential use as PGPB.The country of Chile is long  and narrow (mean width 177 km) and harbors a great variety of pristine ecosystems. The Atacama Desert (AD) is located in the northern region of Chile (from 18°24′S to 29°55′S) and is considered among the driest places on earth. In contrast, the Chilean Patagonia (from 41°08S to 56°30′S) is located in the far south of the country and is a sub Antarctic region. Both regions have extreme environments and their plant-associated bacterial communities have been barely studied thus far. In this context, we have reported that members of the orders e.g., Illumina®, PacBio® and Oxford Nanopore®) have opened new windows into the microbial ecology of a variety of environments, allowing the detailed study of complex bacterial communities in nature as never seen before. Thus, HTS platforms have widely been used to decipher the structure and function of microbiota in different compartments of plants, including as the rhizosphere, endosphere (inner tissues of plants), and phyllosphere 13. Results of 454‒pyrosequencing studies showed that the Proteobacteria (mainly Gammaproteobacteria) were the dominant taxa in the rhizospheres of Atriplex sp. and Stipa sp. (shrubs) grown in the AD14. These authors also postulated that native plants from Chilean extreme environments may attract, select, and conserve specific bacterial groups in order to sustain plant growth and tolerance to local harsh conditions. Based on this supposition, the main goal of the present study was to describe and compare the relative abundances and composition of bacterial communities associated with roots and leaves of plants grown in the AD and PAT regions of Chile by using HTS of 16S rRNA genes.During the last several years, advances in high–throughput DNA sequencing (HTS) technologies  regions of Chile  were used for DNA extraction with Quick‒DNATM Plant/seed Miniprep kits according to manufacturer instructions . The quantity and purity of DNA extracts were determined by measuring absorbance at 260 nm and 280 nm by using a microplate spectrophotometer .Roots and leaves samples were separated and surface sterilized by repeated immersion in 70% (v/v) ethanol for 3 min, followed by 2.5% (v/v) sodium hypochlorite (NaOCl) for 5 min as described by Barra 17. Briefly, PCR conditions were run with an enzyme activation step at 95 °C for 10 min, followed by 40 cycles of 15 s at 95 °C, and 1 min of annealing plus extension at 60 °C. PCR reactions were performed in triplicate per plant species  with 20 µg L−1 of total DNA in a StepOnePlusTM Real–Time PCR System  using PowerUpTM SYBRTM Green Master Mix , by following the manufacturer instructions. The numbers obtained were normalized and analyzed by using one‒way ANOVA, and comparisons were done by using Tukey’s post‒hoc test. Differences were considered to be significant when the P value was ≤0.05.The abundance of endophytic bacteria in each tissue sample was determined by quantitative PCR (qPCR) by using a universal primer set for the bacterial 16S rRNA gene (Bac1369F 5′-CGG TGA ATA CGT TCY CGG-3′) and Prok1492R (5′-GGW TAC CTT GTT ACG ACT-3′) as previously described18 using barcode primers and the dual indexing method. Amplicons were gel purified, pooled, and paired‒end sequenced at a read length of 300 nt on the Illumina MiSeq platform  from UMGC.The distribution and relative abundances of endophytic bacteria in root and leaf tissues was assessed by HTS using triplicate samples of each plant species as follow. The V4 hypervariable region of the 16S rRNA genes were amplified, for bacteria and archaea, by using primer set 515F (5′-GTG CCA GCM GCC GCG GTA A-3′) and 806R (5′-GGA CTA CHV GGG TWT CTA AT-3′). Sequencing was done by the University of Minnesota Genomics Center https://www.mothur.org)19. The first 150 nt were trimmed from sequences to remove low–quality regions at the ends of reads. Fastq–join software was used to join paried–end sequencing reads20, the joined sequencing reads were trimmed to maintain an average quality score >35, a homopolyer length >8 nt. Sequences with >2 mismatches in primer sequences and ambiguous bases were removed. High quality sequencing reads were aligned on the basis of the SILVA database ver. 12321, and subjected to a 2% pre–clustering step to remove possible sequence errors22. The UCHIME software was used to identify and remove probable chimeric sequences23. To avoid the influence of non-microbiota , the sequences were futher filtered by Qiime to remove non-microbiota taxa before a subsequent analysis. Sequence data was rarefied to 700 and 4,500 sequence reads per data set prior to statistical analysis for AD and PAT, respectively. Raw sequencing data were deposited in the Sequence Read Archive (SRA) of NCBI under Accession Number SRP156290.Sequences were analyzed by using mothur program ver. 1.34.0  and permutational multivariate analysis of variance (PERMANOVA). Principal coordinate analysis (PCoA) was performed based on unweighted unifrac distance for the ordination25. The VennDiagram package in R was used to identify shared OTUs of endophytic bacterial communities between root and leave tissues26. Variations in taxa associating with root and leaf tissues were evaluated using linear discriminant analysis (LDA) of effect sizes27, which employs Kruskal–Wallis and Wilconxon rank–abundance tests and then utilizes linear discriminant analysis (LDA) to estimate effect sizes of the features.For statistical analysis, Alpha diversity indices, as well as Good’s coverage, were calculated using the Shannon index and the abundance–based coverage estimate (ACE) through the mothur program. Visualization of the taxonomic distribution of microbial communities was performed using the “ggplot2” package in RG. mucronata and H. pilosella) had greater abundances of bacteria (from 1013 to 1014 16S rRNA gene copies g−1 template DNA), compared with those from AD (D. spicata and P. absinthioides) (from 1010 to 1012 16S rRNA genes copies g−1 template DNA), except roots from G. mucronata (Table P ≤ 0.05) greater abundance of bacteria in both tissues was found in P. absinthioides  compared with those from D. spicata . In PAT plants, a significantly (P ≤ 0.05) greater abundance of bacteria in both tissues was found in H. pilosella  compared with those from G. mucronata .In general terms, plant tissues from Patagonia (obs) were lower in plant tissues from AD compared with those from PAT, ranging in values from 68 to 113 and 208 to 220 in roots, and 103 to 152 and 151 to 160 in leaves, respectively. Similarly, lower ACE values were also observed in AD compared to PAT plants, ranging in values of 148 to 188 and 263 to 314 in roots, and 139 to 211 and 190 to 245 in leaves, respectively. However, and as revealed by the Shannon index, there was significant differences  in bacterial diversity in both tissues of P. absinthioides compared with tissues from D. spicata in AD  compared with PAT (from 98 to 99%) Table . The valProteobacteria (14.88% to 68.53%), Firmicutes (26.03% to 41.59%), Actinobacteria (6.45% to 23.69%), and Bacteroidetes (1.09% to 21.21%) in both tissues  showed large relative abundances of Cyanobacteria, Lentisphaerae and Chloroflexi in roots, and Verrucomicrobia and Fusobacteria and Cyanobacteria in leaves  showed a large relative abundance of Elusimicrobia, Fusobacteria, Spirochaetes and Acidobacteria in roots, and Elusimicrobia, Fusobacteria, TM7 and Verrucomicrobia in leaves , Bacillaceae (24.67%) and Nocardiopsaceae (17.78%) are highlight in roots of D. spicata whereas a higher relative abundance of Halomonadaceae (25.81%) is highlight in leaves of P. absinthioides  and leaves (57.93%) from G. mucronata.With respect to minor taxa, broad taxonomic diversity among samples was found. The tissues of AD plants , followed by Proteobacteria (15) and Actinobacteria (13). In PAT plants, among these 115 shared OTUs, most belonged to the Firmicutes (37), followed by the Actinobacteria (29), Bacteroidetes (25) and Proteobacteria (24). In the PAT, there were a greater number of unique OTUs, relative to those seen at AD, where 305 and 341 and 306 and 195 plant specific OTUs were found in root and leaves from G. mucronata and H. pilosella, respectively , followed by the Proteobacteria (213), Actinobacteria (90) and Bacteroidetes (87). Similarly, in PAT plants, most of unique OTUs belonged to the Firmicutes (512), followed by Proteobacteria (397), Actinobacteria (159) and Bacteroidetes (119) , elucidative served as the keystone taxa in roots of D. spicata, while only the Propionivacteriaceae and Corynebacteriaceae could be used to indicate leaves of D. spicata and P. absinthioides, respectively  of effect size was performed to determine which taxa varied among plant components at two ecosystems. Several bacterial taxa, which belong to the ely Fig. . Moreovetem Fig. . Additio28. However, despite their importance, there is scant knowledge concerning endophytic microbial populations in plants living in extreme environments, including Chile. Our study showed that the abundances of bacteria from the endosphere of root and leaf tissues ranged from 1010 to 1012 and from 1010 to 1014 16S rRNA gene copies g−1 template DNA in AD and PAT, respectively; corresponding from 103 to 107 copies of 16S rRNA genes copies g−1 fresh tissue.Microbial endophytes play a central role in the ecology, evolution, and growth promotion of plants14 16S rRNA gene copies g−1 template DNA of total endophytic prokaryotes were reported in endospheres of olive tree (Olea europaea L.) leaves collected from diverse Mediterranean ecosystems29. Higher abundances than those observed in this current study were reported to be present in the endosphere of rice (107 to 108 16S rRNA gene copies g−1 root) and crops (1010 to 1013 copies of 16S rRNA genes g−1 root) by Ruppel et al.30 and Breidenbach et al.31, respectively. Under stress conditions, Blain et al.32 recently reported abundances of endophytic bacteria from 103 to 105 16S rRNA gene copies g−1 from fresh roots of natural vegetation growing in a hydrocarbon‒contaminated site. In addition, and to our knowledge, there are no studies reporting the abundances of endophytic bacterial populations in plants from Chilean extreme environments. It should be noted, however that a previous study of the rhizosphere of plants from AD and PAT revealed values of 109 and 1011 16S rRNA genes copies g−1 of soil, respectively33.A wide range of endophytic prokaryote densities have been reported in tissues of different plants thus far. Similar to our finding, 1035. However, a significantly greater number of OTUs (from 450 to 3700) have also been reported in plant endospheres by other authors37.With respect to analyses of alpha diversity of endophytic bacterial communities in root and leave tissues, the number of OTUs observed (97% similarity) range from 68 to 152 and from 151 to 220 in the endospheres of plants from AD and PAT, respectively. These values are in accordance with other studies reporting OTUs values from plant endospheres ranging from 100 to 300Paeonia Sect. Moutan) had greater Shannon index values (7 to 9) compared to our study37. In contrast, a recent study reported Shannon index values that were similar to those we found (3 to 4) in root and leaf endospheres of groundsel , also by using the Illumina platform34. Similarly, but by using 454‒pyrosequencing, Correa-Galeote et al.35 reported Shannon index values of 3 to 4 in the root endosphere of maize cultivated at 3,537 meters above sea level in Perú. Interestingly, the Shannon index values found in endosphere tissues from AD plants were generally lower than those reported in other AD habitats, such as soil, lakes and sediments of flat mats, with Shannon index values ranging from 3 to 941. This suggests that endospheres of AD plants may harbor less bacterial diversity compared to other niches in the AD, which is considered oldest and driest place on the Earth42.The Shannon index values ranged from 2.28 to 4.41. and from 2.76 to 4.41 in the endospheres of plants from AD and PAT, respectively. The diversity of bacteria in root and leaf tissues of tree peony  by the Proteobacteria, Firmicute and other phyla  in olive trees, peony and groundsel37. Interestingly, a high relative abundance (31%) of Euryarchaeota was found in roots of D. spicata. Member of Euryarchaeota have also been found colonizing the endospheres of Mediterranean olive trees29 and compartments  of a halophyte plant  from Pakistan44.Our Illumina-based analyses revealed the dominance of members of the phyla Lentisphaerae, Chloroflexi, Verrucomicrobia, Elusimicrobia, Fusobacteria, Spirochaetes and Acidobacteria. Low numbers of sequences of these phyla were observed by Hardoim et al.4, when endophytic data sets from all peer–reviewed publications were revised in the ISI Web of Science and PubMed databases. Similarly, our analyses interestingly revealed the presence of members of the phylum Cyanobacteria in roots and leaves of AD plants. High abundances (24% to 47%) of members of this phyla have also been reported in the endospheres of grasses  and mangrove (Kandelia obovata)45. Lower abundances (1.7%) of Cyanobacteria have also been reported in the endosphere and other compartments (rhizosphere and rhizoplane) of the medicinal perennial plant Stellera chamaejasme L.46. It is noteworthy that Cyanobacteria are a diverse group of photosynthetic bacteria (some of them nitrogen‒fixing) that live in a great variety of extreme environments, including soils and rocks from the AD and hot springs and lakes from Argentine Patagonia48. However, and to our knowledge, there have been no previous reports of Cyanobacteria associated with the endosphere of plants in AD and PAT.Relative to what was seen in tissues from the AD and PAT as minor taxa, both ecosystems had a great diversity, represented by members of the Halobacteriaceae, Bacillaceae, Nocardiosaceae and Halomonadaceae in AD plants, and Pseudomonaceaea in G. mucronata in PAT. Halobacteriaceae and Halomonadaceae are phylogenetically diverse groups of Archaea and Eubacteria able to survive and proliferate in hypersaline habitats, such as soils from AD50, and including some endophytes, such as Euryarchaeota, Kushneria endophytica, Salinicola tamaricis53. Similarly, Bacillaceae and Pseudomonaceae families are commonly found as inhabitant of plant endospheres54. It is noteworthy that the contrast in the family diversity among AD and PAT plants reveals significant differences in the composition of endophytic bacterial communities between locations.At the family level, bioinformatic analyses indicated that there was high diversity in the endosphere of plants from both ecosystems, highlighting some dominant groups as the 55. Similarly, Gadhave et al.56 reported that soil inoculation with Bacillus spp. modified the diversity, evenness, and community composition of endophytic bacterial in roots of broccoli. However, the composition of endophytic bacterial communities might not only to be ruled by biotic factors (such as plant genotype or competition with other microbes), but by abiotic factors including climate (temperature and drought)57 and soil cultivation history35. In this context, significant differences between plant species and locations were also observed in rhizosphere bacterial communities in plants from the AD, PAT and Antarctic14. In addition, differences in endophytic bacterial community composition may be due to a great portion of unique OTUs  compared with the shared OTUs  between the plant species as revealed by diagram Venn. Bacteria belonging to the Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes, were mainly observed among the unique OTUs observed in plants. The low number of shared OTUs, compared with unique OTUs, is in contrasts with observations in tree peony grass and mangrove, where Venn analysis showed overlap patterns of OTUs between endophytic bacteria in root and leaves samples45. Therefore, our results suggest that endosphere of the plant species we studied in each Chilean extreme environment harbored specific bacterial communities unique to their location and plant genotype (species), where Bacillaceae and Enterobacteriacea could serve as keystone taxa as revealed our LDA analysis.A recent study postulated that plant species are able to recruit specific endophytic bacterial communities


The Cmic, Nmic and Pmic values were highest under the PPPR system at both Ellembelle  and Jomoro  sites.Two field experiments were conducted at Ellembelle and Jomoro districts in the Western region of Ghana where rubber cultivation is a predominant farming activity. The objective of the study was to assess the effect of rubber and plantain intercropping systems on selected soil properties. The experiment was arranged in a randomized complete block design (RCBD) with 3 replications. The treatments were the sole crop rubber (R), sole crop plantain (P) and three intercrop systems comprising an additive series of plantain: one row of plantain to one row of rubber (PR), two rows of plantain to one row of rubber (PPR) and three rows of plantain to one row of rubber (PPPR). Generally, agroforestry systems improved the soil hydraulic properties considerably, with the highest cumulative infiltration rates of 5.16 and 8.68 cm/min observed under the PPPR systems at the Ellembelle and Jomoro sites, respectively. Microbial biomass C (C Natural rubber production in Africa is about 5% of the world’s production, with Cote d’lvoire being the lead producer , followed by Liberia. The current production capacity of Ghana, which is 15,000 tonnes per year is anticipated to reach 50,000 tonnes by the year 2020 [The major challenge in the production of rubber worldwide, is the long gestation period (six years), which deprives the farmers of sustainable income during the immature phase, as well as, uneconomic land size  available for plantation establishment . In consDespite the several socio-economic benefits of rubber, such as diversification of agricultural products, increase in income levels for farmers, employment opportunities in the farming communities, foreign exchange earnings, and enhancement of women economic emancipation , there aField trials were conducted at two different locations in the Western region of Ghana where rubber production is prevalent. The locations were Crops Research Institute of the Council for Scientific and Industrial Research (CSIR-CRI) stations at Aiyinasi in the Ellembelle district (between longitudes 2° 05’ W and 2° 35’ W and latitude 4° 40’ N and 5° 20’ N) and Tikobo No. 2—Ehiamadwen in the Jomoro district (between latitudes 4° 80’ N and 5° 21’ N and longitudes 2° 35’ W and 3° 07’ W) . The Ell’ W in the Jomoro district was conducted on an abandoned regenerated oil-palm plantation. The field size was 102 m x 102 m for each experimental set-up. The experiment comprised five treatments: sole crop rubber (R), sole crop plantain (P) and three intercrops consisting of an additive series of plantain, one row of plantain (PR), two rows of plantain (PPR) and three rows of plantain (PPPR) between two rows of rubber arranged in a Randomized Complete Block Design with three replications (The study field at Aiyinasi (05° 03.517’ N and 002° 29.782’ W) in the Ellembelle District was a cleared old and abandoned rubber plantation with regenerated tree species, whereas the trial at Tikobo No. 2 –Ehiamadwen 05° 10.234’ N and 002° 28.768ications .The rubber clone GT1 and the false horn plantain variety  were used for the study. Planting density of rubber was 555 plants/ha in both sole and intercrops, 555 plants/ha for PR, 1111 plants/ha for PPR, 1666 plants/ha for PPPR, and 1666 plants/ha for P. In all intercrop treatments, rubber was planted at a spacing of 3 m within, and 6 m between rows, whereas 3 x 2 m spacing was used for the planting of sole plantain crops. Intra-row spacing for both rubber and plantain was kept constant at 3 m, whilst the inter-row spacing was varied in keeping with the number of plantain rows as 3 m, 2 m and 1.5 m in PR, PPR and PPPR, respectively . Rubber Soil samples were collected at random at 0–15 cm, 15–30 cm and 30–60 cm depths at 10 cores per depth (30 cores per plot) from the plots in each block and were bulked as three composite samples representative of the block for initial characterization before the treatments were imposed. The final soil samples were also taken from each plot at 10 cores per depth (30 cores per plot). These samples were analyzed after they were air–dried, crushed and sieved through a 2 mm sieve for the determination of particle size distribution , bulk deth edition). Mean separation was done using the standard error of difference (SED) at a significance level of 0.05. The Duncan’s Multiple Range Test was used to identify pairs of means that differed significantly.The data obtained from the two field trials were subjected to analysis of variance (ANOVA) using GenStat  in the 0–15 and 15–30 cm depths, whereas, for the Jomoro site, textures ranged from sandy loam (0–15 cm), sandy clay loam (15–30 cm) to clay loam (30–60 cm). Furthermore, hard pan was encountered along the soil profile, especially within the 30–60 cm depth in both sites. This is evidenced by the high clay contents down the profile, especially, at the Jomoro site .Summary of the results on soil hydro-physical properties are presented in Total N was significantly higher (P ≤ 0.05) under the agroforestry PPR (0.22%) and PPPR (0.22%) cropping systems compared to the other cropping systems  at the Ellembelle study site. The lowest total N value (0.18%) was recorded under the sole rubber (R) cropping system. At the Jomoro study site, there was no significant difference (P > 0.05) in the total N under the various cropping systems. The total mean nitrogen content across the various cropping systems at the Ellembelle site was 0.20% while that of the Jomoro site was 0.14% . This trSoil organic carbon SOC was significantly higher (P ≤ 0.05) under the PPR (2.47%) and PPPR (2.71%) cropping systems  than theThe effect of the treatments on the soil available P content at 0–15 cm is shown in The results presented in Soil organic carbon (SOC) differed significantly (P ≤ 0.05) among the various cropping systems at both Ellembelle and Jomoro study sites. At the Ellembelle site, the highest SOC (1.78%) was obtained from PPPR cropping system whiles the lowest was recorded under the sole rubber system. At the Jomoro site, the highest SOC was obtained under the sole plantain cropping system (1.31%) with the lowest (1.0%) from the PR and PPPR treatments. The mean SOC was higher at the Ellembelle site (2.33%) compared to the Jomoro site which has a value of 1.07%.Available soil P was significantly (P ≤ 0.05) influenced by the different cropping systems at the Ellembelle study site. The highest value of 3.84 mg/kg was obtained from the PPPR and the lowest (0.12 mg/kg) from R cropping system. Similar to the observation made in the 0–15 cm depth, results showed that available soil P was not detected at 15–30 cm under the different cropping systems at the Jomoro site. The soil pH was significantly influenced by cropping systems at the Ellembelle but not at the Jomoro site. The highest pH value under the cropping systems at the Ellembelle study site was obtained from the sole plantain treatment (P). The mean pH was similar under the cropping systems at Jomoro than Ellembelle .The total soil N and available P at 30–60 cm depth were not affected by different cropping systems at the Ellembelle and Jomoro study sites . Soil Ormic. The highest soil microbial biomass C (Cmic) was recorded under the PPPR system in both Ellembelle and Jomoro sites. The lowest Cmic were recorded under the sole rubber vegetation in Ellembelle and sole plantain systems in Jomoro. The Cmic increased with increased plantain planting density at both Ellembelle and Jomoro study sites. As with Cmic, soil microbial biomass nitrogen (Nmic) and phosphorus (Pmic) were highest under PPPR in Ellembelle and Jomoro. From the results, Pmic was very low in Jomoro (< 10 mg/kg) compared to Ellembelle.Soil microbial biomass (SMB) C, N and P were significantly affected in both sites under the various cropping systems . PresentThough soil total N and SOC was higher at Ellembelle site compared with Jomoro site, assessment of the crop parameters revealed similar (P > 0.05) productivity at both sites. Plant tissue analysis was conducted on various parts of plantain and rubber to ascertain the concentrations of some macronutrients as a results of the treatments Tables  and 7The results revealed that cropping systems had no significant effect on bulk density for the different sampling depths, however, high bulk density values were observed under the PPPR and R systems in the 0–15 and 15–30 cm depths at Jomoro, and PPPR in both 0–15 and 15–30 cm depths at Ellembelle . The relThe results showed that cropping systems had no significant effect on bulk density . This coThe different rubber and plantain cropping system did not significantly influence soil moisture content (SMC) at both sites in the 0–15 cm depth, however, significant differences were observed in the 15–30 cm depth . This co1/2), which controls the initial stage of infiltration , and the steady state infiltrability (2.12 cm/min), which describes the infiltration rate toward saturation of the soil. The high water penetration rate in the soil observed under the PPPR, PPR and PR systems are clear indications that the rubber in agroforestry systems have considerable effects on both bio-physical and chemical processes that affect soil health [Generally, the plantain based cropping systems promoted the movement of water in the soil as evidenced by the high cumulative infiltration amount, infiltration rate, sorptivity and steady state infiltrability values at both study sites . This iml health . Thus, tl health , 28, whil health .From the study, the intercropping systems  generally resulted in increased N and SOC contents than the sole cropping systems (R and P). These findings are in agreement with Barua and Haque , who repComparing the individual agroforestry systems, the soil pH in the different depths did not differ from the initial values in both study sites. The differences in pH observed at different soil depth at both sites could be as a result of leaching and lack of mixing of the soil profile, and variations in the soil fractions and SOC contents at different depths . The relmic and Nmic were significantly variable among the various treatments at both sites [mic differed significantly across the intercropping systems at both Ellembelle and Jomoro study sites. However, compared with other studies, such as Vance et al. [mic under the different cropping systems in both sites could be attributed to variable microclimates resulting from the differences in vegetation cover and actively growing vegetation [mic under vegetation could create and enhance soil physical structure with a concomitant effect on carbon sequestration. However, the buildup of Cmic in the PPPR systems could subsequently result in increased fluxes of trace gases from microbial processes [The agroforestry systems had significant effect on soil microbial biomass C, N and P among in both the Ellembelle and Jomoro sites . Consistth sites , 41, 42.e et al.  for tempe et al.  for Amaze et al.  for humigetation , 45, espgetation  reportedrocesses , 46, 47.mic and Pmic in soils. The PPPR systems in both sites had the highest Nmic, which implies a corresponding higher nitrification rate than the other cropping systems [mic was observed under PPPR systems in both study sites. This suggests the potential contribution of effective agroforestry systems in soil microbial buildup. The observed high soil microbial biomass (SMB) in the 0–15 cm depth supports the findings of higher microbial biomass in the upper soil layer [mic under wheat crop rows in an intercropping system in China. Generally, differences in litter quantity and quality in these systems could have resulted in the significant differences in the soil microbial biomass. The high SMB associated with plantain fields could be attributed to the high litter production by plantain than rubber.The results showed that increasing vegetation cover through increased plantain population influenced both N systems . Accordi systems , this coil layer , 49. Yanil layer , reportemic/SOC ratio) [mic/SOC ratio was used in this study as an indicator of the efficiency in the utilization of organic substrates by microbes [mic/SOC ratio can be significantly enhanced by SOM management, improving soil microbial characteristics and slowly increasing SOC [mic/SOC ratio can be effectively used as early signs of soil quality degradation. Amongst the various cropping systems, the lowest value was found under sole rubber (R) at both sites, indicating that the size of Cmic as a proportion of the total SOC is greater under the plantain vegetated systems , with PPPR having the highest. The Cmic/SOC ratios under the various cropping systems at both study sites were generally low when compared to the critical threshold of 2.0 [The amount of metabolic active C in the total soil organic matter (SOM) as a result of the various cropping systems was described using the microbial quotient (CC ratio) ; 51–53. microbes , 54, andmicrobes . From thmicrobes . Thus, tsing SOC . Hence, sing SOC  and Wardsing SOC  reportedd of 2.0 , 54, whid of 2.0 .mic/Nmic ratios estimated were used as indicator of N supply ability, and also to describe the structure and state of the microbial community under the different intercropping systems [mic/Nmic ratios of 5.2 in an arable land, and 20.8 in a forest soil. Djagbletey [mic/Nmic ratios ranging from 5.9 to 17.7 under three different forest covers in a Savanna ecosystem in Ghana. In their study, Logah et al. [mic/Nmic ratios ranging from 3.9 to 35 in an agricultural land in the semi-deciduous forest zone of Ghana. The low ratios observed in this study are a clear evidence of the low N contents [mic/Nmic ratios observed in this study could also be attributed to the substrate imbalance owing the presence of woody plants (rubber trees), which have extremely high C/N ratio, up to about 400 [The soil C systems . Compare systems  reportedagbletey  also reph et al.  also foucontents , 63. Acccontents , the C/Ncontents . The lowbout 400 –67.mic and Nmic, levels Pmic also increased with decreasing plantain density. Evidently, with Cmic, Nmic and Pmic being significantly higher under the plantain-based systems suggests that distribution pattern of soil microbial biomass under the different cropping systems was likely governed by plantain vegetation density. Microbial quotient and microbial biomass C/N ratio were efficient in the determination of the differences among various intercropping systems in the study. The results would contribute to the knowledge on flow of carbon and energy through soil microbial biomass under different agricultural land use systems.High levels of microbial biomass C and N were observed in the high plantain density plots. Similar to CWith regard to the soil hydro-physical properties, greater improvements were observed under the rubber-plantain intercropping systems than the monocropping systems. Additionally, the study has revealed that plantain has the ability to improve soil physical and hydraulic parameters through surface cover by litter fall and canopy cover, and also root coverage in the soil. This study has revealed the potential of agroforestry under rubber to present an opportunity in increasing land productivity and improve soil fertility. In addition, agroforestry practices can increase the nutrient cycling, control of surface runoff and/or erosion, and C sequestration.S1 Data(RAR)Click here for additional data file.


There is a need for novel mechanical devices for dispatching poultry on farm following the introduction of EU Regulation (EC) no. 1099/2009 On the Protection of Animals at the Time of Killing. We examined three novel mechanical killing devices: Modified Armadillo, Modified Rabbit Zinger, a novel mechanical cervical dislocation device; and traditional manual cervical dislocation. The four killing methods were tested on 230 chickens across four batches. We measured behavioural, electroencephalogram and post-mortem outcomes in anesthetized laying hens and broilers at two life stages (juveniles and adults/slaughter age). Graeco Latin-Square designs systematically randomized killing treatment, bird type, age and kill order. All birds were lightly anaesthetized immediately prior to the killing treatment with inhalation of Sevoflurane. The novel mechanical cervical dislocation method had the highest kill success rate  of a mechanical method (96%). The Modified Armadillo was the least reliable with 49% kill success. Spectral analysis of electroencephalogram signals at 2 s intervals for successfully killed birds only revealed progressive decreases in median frequency alongside increases in total power. Later, total power decreased as the birds exhibited isoelectric electroencephalogram signal. Latencies to pre-defined spectral ranges associated with unconsciousness showed that birds subjected to manual and novel mechanical cervical dislocation achieved these states sooner than birds subjected to the modified Armadillo. Nevertheless all methods exhibited short latencies (<4 s). The Modified Rabbit Zinger had the shortest duration of reflex persistence for nictitating membrane, pupillary and rhythmic breathing post method application. Of the methods tested, the novel mechanical cervical dislocation device is the most promising mechanical method for killing poultry on-farm based on a range of behavioural, electroencephalogram and anatomical parameters. This device has the potential to fulfil the current need for a mechanical alternative to manual cervical dislocation. Poultry need to be dispatched on-farm for multiple reasons , either on a group/large scale (e.g. whole-house or containerized gassing –2) or asDetermination of time to loss of consciousness is crucial when ascertaining the welfare impact of a killing method. EEG  analysis is a useful tool which has been applied for this purpose in various livestock species –15. The In the field, it is not practical to record EEG in each animal to confirm unconsciousness and death , therefoThe aim of this experiment was to evaluate the efficacy and welfare impact of three novel killing methods for on-farm killing of poultry using both iEEG and reflex measurements. The methods tested were the modified Armadillo (MARM), a puntilla-style device; modified Rabbit Zinger (MZIN), a penetrating captive bolt; and a novel mechanical cervical dislocation glove (NMCD), which aids the manual technique. These three killing treatments had previously been trialed with cadavers , where tThis work was performed under Home Office (UK) authority via Project and Personal Licenses and underwent review and approval by Scotland’s Rural College’s (SRUC) Animal Welfare and Ethical Review Body (AU AE 34–2012).ad libitum food and water, wood-shavings litter and kept at lower than commercial stocking densities  and with suitable environmental enrichment .A total of 232 female laying hens and broiler chickens, each at two different ages, were used for the study . Graeco Broiler chicks, which were not implanted with iEEG electrodes due to their small skull size, were housed in one pen as a group (L 1.5 m x W 2.5 m x H 1.5 m). Slaughter-age broilers, layer pullets and layer hens were kept singly  following iEEG electrode implantation surgery. Following the completion of batch 1, it became apparent that the presence of the iEEG electrode was significantly hampering the performance of one of the killing methods, the modified Zinger, and subsequently the birds in the batches assigned to the Zinger treatment (batches 2–4) did not undergo iEEG implantation surgery.Prior to the predetermined killing days across all four batches, two birds were euthanised on welfare grounds, reducing the total N for two treatments by one bird each .Only layer pullets, laying hens and slaughter-age broilers were implanted with iEEG electrodes. Five days prior to the designated individual bird killing date across the four batches, a total of 109 birds underwent surgery to implant iEEG electrodes under general anesthesia, induced and maintained with sevoflurane . All birds received a pre-medication of dexmedetomidine , approximately 30 minutes prior to surgery. At the start of surgery, carprofen  analgesic was administered to provide post-operative pain relief. The bipolar iEEG implantation approach has been described previously ,22. BrieAll killing methods applied are discussed in detail in Martin et al  and wereThe Armadillo is a brain-stem penetration device designed to dispatch game birds in the field ,8. The dThe Rabbit Zinger  is a penThe novel manual cervical dislocation device (NMCD) was designed to create a mechanical method which mirrored the technique of the manual cervical dislocation method for poultry ,8. BriefTraditional manual cervical dislocation was included in the experiment as a control. This was performed by an experienced operator following the HSA’s guidelines .Across the four batches, Graeco Latin-Square designs were used to systematically randomize killing treatment, bird type and age and kill order. Killing treatment was allocated to individual birds so as not to confound killing treatment with pens. Birds were killed over four days for each batch, with 10–18 birds killed per day. All birds were brought to the experiment room (separate to their housing environment) in individual pet carriers. Immediately before each killing treatment application, iEEG implanted birds were fitted with instrumentation: a reusable custom-made Lycra harness was secured using Velcro fastenings behind the bird's head and incorporated a pocket positioned on the bird's back which contained a custom-made telemetry/logging device, capable of logging simultaneous iEEG and ECG signals and described elsewhere –23. BrieTwo ‘physiological waveform’ input channels were used to record ECG and iEEG (sampling rate 1000 Hz). Logging was triggered and stopped with an external switch and logged data were recorded onto industry-standard ‘micro-SD’ memory cards . Two identical loggers were alternated. The logger harness was additionally secured to the birds with elastic bandage . All birds were lightly anaesthetized immediately prior to the killing treatment being applied via a “fast knockdown” method using a face mask , at an 8% concentration vaporized in 100% oxygen for a maximum of 20 seconds). Birds were confirmed as non-responsive to pain immediately prior to killing treatment application by applying a sharp toe and comb pinch. The approach used represented a balance between ensuring that birds were unconscious when killed but minimizing ongoing effects of anesthesia on the iEEG after killing. Light anesthesia as induced by this method is transient, and birds readily recover , so if not killed the birds would have rapidly regained consciousness. All killing methods were applied by a single fully trained operator, holding a valid Welfare of Animals at the Time of Killing (WATOK) license for killing operations outside a slaughterhouse.Kill success was defined as only one application attempt with no signs of recovery (e.g. return of rhythmic breathing). If any signs of recovery continued for 15 s  the bird was emergency euthanised; the method of euthanasia was killing treatment dependent in order to prevent post mortem examination data being voided  gunpowder cartridge); for MZIN and MARM it was by manual cervical dislocation.Seven reflexes and behavior  were assThe logged data files were uploaded into a data acquisition and analysis program . iEEG activity was sampled continuously in 2 s epochs during a two minute baseline period (awake bird), during “fast knockdown” with anesthetic (15–20 s), at killing method application and post-kill activity until all behaviors and reflexes had ceased for a minimum of 30 s. Visual inspection was used to eliminate obvious movement artefacts which rendered the signal meaningless, while epochs that were apparently affected by minor electrical noise interference were subject to post hoc ‘filtering’ using the data interpolation technique . The iEEThree epochs (midpoint ± 10 s either side) of iEEG wave activity were obtained and analyzed in the 2-min baseline period (conscious bird). One epoch was analyzed during the “fast knockdown” period. Overlapping 2 s epochs were obtained from -2 s to +5 s  relative to the time of killing treatment application (estimated kill application time = 0 s). From +5 s to +59 s, a continuous series of non-overlapping 2 s epochs were analyzed. Thereafter 2 s epochs were sampled from the midpoint every 15 s, until three consecutive samples were judged to be isoelectric.th Edition, Rothamsted Research, UK) programs: total power (PTOT), defined as the total area under the power spectrum curve [Three spectral variables were calculated with coded Genstat  from the analysis for calculating latencies to unconsciousness ranges (F50 ≤ 12.7 Hz and ≤ 6.8 Hz) ,22. TherPost-mortem examination was carried out immediately after confirmation of death. Specific post-mortem measures were recorded for each killing treatment as their target areas were different. For all killing treatments binary yes/no measures were recorded for broken skin, external blood loss and subcutaneous hematoma. For the MZIN and MARM, seven specific measures were recorded: skull penetration location; binary measures of damage to the left forebrain, right forebrain, cerebellum, midbrain and brainstem; and the presence/absence of an internal brain cavity hematoma. For killing treatments which caused trauma to the neck of the bird, seven specific post-mortem measures were assessed: four binary measures were recorded for dislocation of the neck, vertebra damage (e.g. intra-vertebra dislocation/break), damage to neck muscle, and whether the spinal cord was severed. The level of cervical dislocation was recorded , as well as the gap length (cm) between the dislocated cervical vertebra. The number of carotid arteries severed was also recorded as either zero, one or both.Device success was defined as when the device caused the expected anatomical damage, as well as producing sufficient damage which would be expected to result in rapid death. Device success criteria were device specific: MARM–back of skull was penetrated and brain stem severed; MZIN–skull was penetrated and severe damage to a minimum of one gross area of the brain; for both manual cervical dislocation and NMCD–complete dislocation at C0-C1, with severance of the spinal cord and both carotid arteries, no breakage of the skin or sign of crushing injury to the trachea or esophagus.th Edition). Statistical significance was termed by a threshold of 5% probability based on F tests. A p value ranging from >0.05 - <0.10 was defined as a statistical trend. Summary graphs and statistics were produced at the bird level. Statistical comparisons for kill success and device success were conducted via Generalized Linear Mixed Models (GLMMs), using the logit link function and binomial distribution.All data collected at the bird level were summarized in Microsoft Excel (2010) spreadsheets and analyzed using Genstat  and head damage methods (MZIN and MARM) and analyzed separately. Statistical comparisons were performed on sub-sets of data to remove failure birds (i.e. kill success “no”) in order to prevent data skewing. All post-mortem binary measures (e.g. skin break yes/no) and categorized measures (e.g. brain damage grade) were analyzed via GLMMs using logit link function and binomial distribution. Device success was used as a fixed effect within all the models.For the reflex/behavior durations, statistical comparisons were performed on successfully killed birds only, in order to prevent data skewing. The presence/absence of each reflex and behaviour was summarized into interval counts (e.g. present in 0–15 s = 1 count), therefore summarizing the data into means of the maximum interval counts at the bird level for each reflex, which were then converted back into the time dimension (s). GLMMs with logit link function and Poisson distributed errors were fitted to the interval counts. Overall statistical comparisons across the killing treatments were conducted. Further analysis involved sub-setting the data into two groups: (1) NMCD and MCD; and (2) MZIN and MARM, which allowed post-mortem effects to be fitted into the models as factors. Device success was used as a fixed effect within all the models.For all models the random effects included the batch and the kill day. All fixed effects were treated as factors and classed as categorical classifications and all interactions between factors were included in maximal models.F3,229 = 24.46,p < 0.001) with the MCD being the most successful method, (100.0% kill success), followed by NMCD (96.0%), MZIN (75.0%) and MARM (48.7%). Bird type, age, iEEG implantation status , body weight, kill order and all interactions did not affect kill success. Device success  was significantly affected by killing treatment , with the MZIN being the most successful (75.0%), matching its kill success. The NMCD, MCD and MARM all had less than 45% device success. Device success was also affected by bird age , being more likely to be achieved in both younger birds (69.9 ± 0.1%) compared to the two groups of older birds (58.9 ± 0.1%). There was no effect of bird weight, bird type, iEEG implantation status or kill order on device success.A total of 33 birds were not killed at the first attempt across the killing treatments: MARM = 19/39 birds; MZIN = 30/40 birds; NMCD = 72/75 birds; and MCD = 0/76 birds. The 33 birds were immediately killed with an emergency method, thus invalidating their reflex/behaviour, iEEG, ECG and post-mortem data. Kill success (defined as only one application attempt with no signs of recovery) was therefore affected by killing treatment , F50 (mean = 23.0 ± 1.3 Hz) and F95 (mean = 70.7 ± 1.5 Hz),were not significantly different between killing treatments. There were also no significant differences between killing treatments for PTOT (mean = 4001.3 ± 1016.6 μV2), F50 (mean = 10.2 ± 0.5 Hz) and F95 (mean = 24.2 ± 2.2 Hz) during the knock-down (unconscious/light anesthetic) phase before killing methods were applied.In the baseline period (awake/conscious) the spectral variables of PTOT  reduced the time to F50 < 12.7 Hz . Bird type  also had an effect, with layer hens (mean = 5.3 ± 1.6 s) exhibiting longer F50 < 12.7 Hz timings than slaughter-age broilers (means = 2.4 ± 0.7 s). The remaining fixed effects  were not significant.Killing treatment affected the latency to F50 < 12.7 Hz . The MCDF2,35 = 4.24,p = 0.022), which were not significantly different. Mean latency to F50 < 6.8 Hz was significantly shorter when the device application was optimal ). Bird type/age also had a significant effect  with shorter latencies for slaughter-age broilers (mean = 4.7 ± 2.1 s) compared to layer type birds (hens and pullets) (mean = 5.7 ± 1.5 s).Killing treatment also had an effect on the first time to F50 < 6.8 Hz, with the MARM having longer latencies to F50 < 6.8 Hz compared to NMCD and the MCD  and last  isoelectric EEG occurrence, with the MARM having the longest latencies compared to the MCD and NMCD.The first time to isoelectric and last time not isoelectric were calculated in order to provide an estimate of when brain death occurred. Killing treatment had an effect on latencies to both first ; nictitating membrane reflex ; pupillary reflex  and rhythmic breathing   and rhythmic breathing durations , with heavier birds displaying longer durations of both reflexes. Bird type had an effect on the pupillary reflex , wing flapping , leg paddling  and cloacal movement  durations post method application, with broilers exhibiting significantly shorter durations.Killing treatment had a significant effect on observed durations of jaw tone (= 0.036) , with thF1,152 = 0.05,p = 0.816), with the majority receiving a C0-C1 dislocation . MCD resulted in the lowest dislocation level recorded (C4-C5), which occurred in two birds, a layer hen and a slaughter-age broiler. Age at killing  and neck gap  affected dislocation level, with older birds being more likely to have a lower dislocation than younger birds. Neck gap was affected by killing treatment , with NMCD (mean = 6.6 ± 0.3 cm) resulting in larger neck gap sizes compared to the MCD (mean = 5.3 ± 0.3 cm). Bird type  and bird age  also had an effect with layers and younger birds having larger neck gap sizes compared to broilers and adults . With NMCD, 23.6% of birds exhibited breakage/tearing of the neck skin, compared to 13.2% of MCD birds, although this difference was not significant. For both methods, almost all birds sustained a subcutaneous haematoma  and severe muscle damage and tearing to the muscle in the neck . Most birds also had one or both carotid arteries severed , with NMCD more likely to sever one or both arteries than the MCD . Larger neck gaps were more likely to result in one or both carotid arteries being severed ).For successfully killed birds from cervical dislocation methods, all had their necks fully dislocated and their spinal cord severed, with no intra-vertebrae damage, irrespective of cervical dislocation method. The location of the dislocation point did not differ between the two methods  and killing treatment . For successfully killed birds, the range of skull areas damaged was lower in both devices compared to unsuccessfully killed birds. When the MARM was successful, skull damage occurred only around the rear central area of the skull , however, when unsuccessful the areas of damage were more varied. When successful, the MZIN damaged the middle and rear areas of the skull, again, when unsuccessful the areas of damage varied. For both devices, in some unsuccessful kills, no damage to the skull was observed, with only soft tissue damage around the head and neck .Kill success had a significant effect on a number of post-mortem measures for both brain-trauma killing treatments . Damage F1,48 = 5.50,p = 0.022) and brain stem  were damaged .In successfully killed birds, the brain damage occurred in the cerebellum and brain stem after application of the MARM, while for the MZIN all brain regions except the brain stem were subject to damage. Damage to all brain regions except for the cerebellum was affected by treatment. Bird weight, irrespective of kill method, affected whether or not the right forebrain  and traditional manual cervical dislocation on laying hens and broilers at two ages. We captured iEEG signals and detailed behavioural/reflex responses, as well as post-mortem anatomical effects in the same individual birds, during application of on-farm dispatching methods. Manual cervical dislocation was the most reliable (100% success) followed by the NMCD (96%). This is the first assessment of true manual cervical dislocation  our findNMCD was easy to use and adaptable to different bird types and ages, although it was apparently limited by bird weight, as the three birds that weighed greater than 3.3 kg were not killed on the first attempt. This may have been due to the operator’s strength rather than the device itself and the same limitation may have applied to the MCD treatment had any of the birds in this group exceeded 3 kg (they did not). The reliability of NMCD concurs with reported high kill success rates of other mechanical cervical dislocation devices (e.g. Burdizzo and killing cone ).The lower success rates observed when using MARM and MZIN devices were primarily due to difficultly in aiming the devices. The top heaviness of the MZIN as well as the small size of the bird’s head in relation to the bolt muzzle made it difficult to aim and balance the device on the head prior to firing. As the device was originally designed to kill rabbits, the significant difference in size (and ratio of head size to bolt muzzle) and shape of the skull of the target species meant that, despite the modifications made, the device was not reliable. Other captive bolt devices  have been more successful, with 100% kill success in poultry ,13.The MThe use of iEEG and behavioural recordings in this work to infer level of unconsciousness post device application had limitations, since the birds were anesthetized immediately prior to testing. However, we attempted to reduce ongoing anesthetic effects by using a rapid induction method which has shown in previous research to have minimal effect (i.e. rapid anesthetic recovery times) on the iEEG pattern –28 and rWelfare impact was inferred in three ways: duration of reflexes (e.g. jaw/mandible tone), latencies to onset of particular iEEG frequency thresholds , and latencies to onset of previously validated unconsciousness thresholds. The baseline (awake) iEEG parameters concurred with the mean spectral variables reported in previous research . As in pThe MCD method resulted in the shortest time to F50 < 12.7 Hz (2.6 s). However the mean latency to onset of permanent electrical brain activity below either threshold  was greater compared to first onset, demonstrating that the level of unconsciousness varied, but was below the sedation threshold  and was Available iEEG data did provide an insight into the time to brain death (i.e. when the trace became isoelectric) for the MARM, NMCD and MCD. NMCD resulted in the shortest duration to first time isoelectric and last time not isoelectric compared to the only other mechanical device (MARM), though the difference between NMCD and MCD was not significant. The shorter duration to brain death may not have welfare benefits, but since operators must confirm the success of a kill immediately post-application and must not move on or kill another individual until the present one is confirmed dead , the shorter the duration to brain death, the quicker these measures will cease and the operator can continue with their duties. This could indirectly benefit to bird welfare, as if operators are forced to wait for less time, they may be more likely to wait and confirm death, reducing the possibility that a severely injured bird would be left unattended for a prolonged period of time.The duration of reflexes, the loss of which are considered to indicate death  did Birds showed convulsive behaviors (e.g. leg paddling and wing flapping) post treatment application, and these behaviors continued beyond iEEG activity. This was true for all bird types, ages, and with all killing treatments. Similar results have been identified in previous research and confirm that the behaviors are not treatment specific ,5,12,21.As with reflexes and iEEG indicators of death, there was no correlation between reflexes and iEEG data for consciousness, highlighting a concern that established consciousness indicators for poultry as well as other species may not be accurate. However, the lack of relationship appears to be always in the same direction, with reflex durations being greater than the iEEG unconsciousness thresholds. When considering both measurements of reflex/behaviour durations and iEEG analysis (e.g. latencies and time series), the NMCD device appears to be the most humane mechanical method with no significant differences to MCD in terms of durations to unconsciousness. Despite the lack of iEEG data for the MZIN, the reflex data suggests it could be considered to be humane, as cranial reflex durations , and jaw tone were abolished quickly post device application . Therefore, if the reflexes are considered highly conservative compared to iEEG measurements, then hypothetically this treatment could be suggested to be the most humane, matching previous findings for captive bolt devices ,11–12. HFor the two most promising methods, MCD and NMCD, all successfully killed birds had their cervical vertebrae dislocated and spinal cords severed. The location of the dislocation was very consistent, with approximately 80% of birds receiving a C0-C1 dislocation. This focusses the trauma around the brain stem and top of the spinal cord, resulting in functional impairment and increased likelihood of neurogenic shock . The NMCAdditional factors which at times affected the reliability, humaneness and consistency of all killing methods were bird type, bird weight and bird age. These were sometimes confounded resulting in interactions which could not be disentangled. The MARM and MZIN devices were most affected by these factors, demonstrating the limited ability of such devices to adapt to individual bird variation. The NMCD and MCD were minimally affected and were able to adapt to different sizes, weights and types of birds. This seems to be primarily due to the operator’s input into the application of the killing method, such that the methods could be subtly adjusted immediately , although this is reliant on the operator’s experience and training to make an assessment of what adjustments were required.In conclusion, our evaluation of three mechanical killing devices and MCD with regard to their reliability, humaneness and consistency demonstrated that NMCD was the most successful novel method. The MZIN did show promise in terms of humaneness with the shortest reflex durations but this was countered by low reliability. The MARM device performed poorly in all three areas demonstrating its lack of suitability as a humane killing method for poultry on-farm. MCD method performed well as a killing method for poultry and matched the performance of NMCD. Collectively, the findings of this study provide evidence that the NMCD is a promising device for killing poultry on-farm, potentially fulfilling a need for a mechanical alternative to manual cervical dislocation.