extfieldsofstudy: Medicine Biology

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CRISPR/Cas9-mediated mutation of OsSWEET14 in rice cv. Zhonghua11 confers resistance to Xanthomonas oryzae pv. oryzae without yield penalty

Background Bacterial blight of rice, caused by Xanthomonas oryzae pv. oryzae (Xoo), is a devastating rice disease in Southeast Asia and West Africa. OsSWEET14, encoding a sugar transporter, is known to be a major susceptible gene of bacterial blight targeted by four different transcription activator-like (TAL) effectors from either Asian or African Xoo strains. However, the OsSWEET14 single knockout or promoter mutants in the Kitaake background are moderately resistant or even susceptible to African Xoo strains. Therefore, in this study, we knocked out OsSWEET14 in rice cv. Zhonghua 11 background for disease assessment. Results In this study, CRISPR/Cas9 was utilized to disrupt the function of OsSWEET14 by modifying its corresponding coding region in the genome of rice cv. Zhonghua 11 (CR-S14). In total, we obtained nine different OsSWEET14-mutant alleles. Besides conferring broad-spectrum resistance to Asian Xoo strains, tested mutant alleles also showed strong resistance to African Xoo strain AXO1947. Moreover, the expression of OsSWEET14 was detected in vascular tissues, including the stem, leaf sheath, leaf blade and root. The disruption of OsSWEET14 led to increased plant height without a reduction in yield. Conclusions Disruption of OsSWEET14 in the Zhonghua 11 background is able to confer strong resistance to African Xoo strain AXO1947 and Asian Xoo strain PXO86. CR-S14 has normal reproductive growth and enhanced plant height under normal growth conditions. These results imply that CR-S14 may serve as a better tester line than sweet14 single-knockout mutant in the Kitaake background for the diagnostic kit for rice blight resistance. The genetic background and increased plant height need to be taken into consideration when utilizing OsSWEET14 for resistant rice breeding.

Background
Bacterial blight of rice, caused by Xanthomonas oryzae pv. oryzae (Xoo), is a devastating rice disease in Southeast Asia and West Africa [1,2]. The pathogen contains type III effectors that can be injected into rice cells directly via the type III secretion system [3]. Transcription activator-like (TAL) effectors, which are the major virulent effectors in Xoo, function like eukaryotic transcription factors to induce target gene expression via binding to the effectorbinding elements (EBEs) in the promoter of the target genes [3,4]. TAL effectors are composed of an N-terminal type III secretion signal, a C-terminal nuclear-localization signal and activation domain and a central repeat domain. The central repeat region consists of 1.5-33.5 tandem repeats that are typically 33-35 amino acids long, and amino acids at the 12th and 13th positions in each repeat are called repeat variable diresidues (RVDs) [5,6]. The number and order of the RVDs determine the recognition specificity of TAL effectors [5,6].
Since Xoo activates OsSWEET14 by binding to the specific EBEs in the promoter region, great efforts were invested into generating resistant rice plants by genetic editing of the promoter region of OsSWEET14 or by identifying natural EBE-mutant alleles in germplasm reservoir for resistant rice breeding [14,15]. The recessive resistance (R) gene xa41(t), which is the natural EBEmutational allele of OsSWEET14, has been identified in African rice varieties [16]. xa41(t) has an 18 bp deletion in the promoter region overlapping with AvrXa7, PthXo3 and Tal5 EBEs, so it confers resistance to Xoo depending on AvrXa7 and PthXo3 for virulence [16]. Since the TalC binding site in the promoter region of xa41(t) is intact, and all the sequenced African Xoo strains harbor the TalC effector, xa41(t) is unable to confer resistance to African Xoo strains [13,16]. Genetically modified rice plants with altered EBEs in the promoter region of OsSWEET14 showed resistance to Xoo depending on the corresponding TAL effector and had normal development [15,17]. However, a previous study found that mutations in the TalC EBE in the promoter of OsSWEET14 resulted in a susceptible response to African strain BAI3 that depended on TalC for virulence in the Kitaake background [17]. Recently, researchers found that a mutation in the TalC EBE alone in the Kitaake background still could not confer resistance to African strains; instead a quintuple-mutant promoter lines (rice with mutated PthXo1, PthXo2, TalC, AvrXa7 and Tal5 EBEs) in the Kitaake background were moderately resistant to African strain AXO1947 [13]. Moreover, the sweet14 single-knockout mutant in the Kitaake background was also susceptible to African strain AXO1947, with a median lesion length of approximately 10 cm, whereas the sweet13;sweet14 double-knockout mutant showed complete resistance to African strains [18]. All of these previous studies demonstrated that single knockout of OsSWEET14 in the Kitaake background was unable to confer resistance to African strains. However, OsSWEET11 and OsSWEET13 are not target genes of African Xoo strains. Theoretically, the mutation of those two genes should not contribute to the resistance response of Kitaake to African Xoo strains. The disease response of different mutants cannot be explained with current knowledge.
OsSWEET14, encoding a sugar transporter with seven transmembrane helices, targets the plasma membrane and is mainly responsible for sucrose and glucose transportation [19,20]. TAL effectors function in diverting the nutritional resources from rice by inducing the expression of OsSWEET14 [19][20][21][22][23][24]. The SWEET11 and SWEET12 single mutant of Arabidopsis did not show obvious morphological defects [20]. However, the double mutant was smaller and had an impaired ability to export sucrose from the leaves [20]. Although the rice OsS-WEET14 T-DNA insertion mutant showed AvrXa7-and PthXo3-specific recessive resistance, the homozygous mutant had dramatic development defects including small seeds and severe growth retardation [9]. The homozygous plants required~30 more days to reach the size of 14-day-old normal plants [9].
In our study, we used CRISPR/Cas9 to mutate the coding region of OsSWEET14 in rice cv. Zhonghua 11 (CR-S14) in order to test whether the disruption of OsS-WEET14 in the Zhonghua 11 background will result in broad-spectrum resistance to Xoo strains including those originate from Africa. This will help us to know whether CR-S14 is a better tester line than sweet14 single-knockout mutant in the Kitaake background for the diagnostic kit for rice blight resistance and also whether the genetic background needs to be taken into consideration when utilizing OsSWEET14 for resistant rice breeding. Moreover, the assessment of CR-S14 agronomic traits will help us understand the role of OsS-WEET14 in development and will provide more information when utilizing the OsSWEET14 knockout mutant for resistant rice breeding.

Results
Generation of rice lines edited in the OsSWEET14 coding region (CR-S14) To modify OsSWEET14 in rice cv. Zhonghua 11, a CRISPR/Cas9 construct targeting two targets in the corresponding coding region of the OsSWEET14 genome sequence was built and transformed into Zhonghua 11 background. Target I and Target II were located in the 1st and 3rd exon, respectively (Fig. 1a). Polymerase chain reaction (PCR) and sequencing were used to detect the modifications in the rice transformants. Two rice lines, CR-S14-2 and CR-S14-6, harbored homozygous mutant alleles in the T 0 generation (Fig. 1b), and the other four lines contained biallelic mutant alleles in the T 0 generation, i.e., CR-S14-1, CR-S14-9, CR-S14-10 and CR-S14-29; homozygous mutant alleles were obtained in the T 1 or T 2 generation. According to the sequencing result, the modification in mutant allele CR-S14-1-I was the same as that in CR-S14-6, so a total of nine heritable mutant alleles were obtained (Fig. 1b).  1 Genetic modification of OsSWEET14 using CRISPR/Cas9. a Two targets were selected in the coding region of OsSWEET14. Target I is located in the 1st exon, while target II is located in the 3rd exon. Two pairs of primers (a and b, c and d) were used to amplify the two target regions for sequencing. Black boxes and white boxes represent the corresponding CDS and untranslated region of the OsSWEET14 transcript in the genome, respectively. a, Target-TalC-F; b, Target-TalC-R; c, Target-S14E3-F; and d, Target-S14E3-R. b Sequences of OsSWEET14 mutant alleles in the target regions. Mutations in CR-S14-2 and CR-S14-6 were homozygous in the T 0 generation, while mutations in CR-S14-1, CR-S14-9, CR-S14-10 and CR-S14-29 were biallelic in the T 0 generation. All the biallelic mutations were homozygous in the T 1 or T 2 generation. CR-S14-1-I was the same as CR-S14-6. Deletions and insertions are indicated by dashes and red letters, respectively. Target I and Target II are underlined in the wildtype (WT). The number of nucleotides that were deleted or inserted in mutant alleles is indicated on the right side of the mutant allele sequence Six of the mutant alleles contained frameshift mutations, i.e., CR-S14-1-II, CR-S14-2, CR-S14-6, CR-S14-9-I, CR-S14-9-II and CR-S14-29-I. The three other mutant alleles, CR-S14-10-I, CR-S14-10-II and CR-S14-29-II, contained in-frame mutations (Fig. 1b). Mutations were also amplified and confirmed in several mutant transcripts (Additional file 1). Since OsSWEET14 is a sugar transporter containing seven transmembrane helices, the function of the protein depends strongly on the helices. The TMHMM2.0 program was utilized to predict the transmembrane helices of proteins encoded by mutant alleles [25]. All of the frameshift mutant alleles encoded proteins with only one or even no transmembrane helices, while all the in-frame mutant alleles encoded proteins containing five or six transmembrane helices (Additional files 2 and 3). Loss of the transmembrane helices had high possibilities of disrupting the transporter activity. This may indicate that all the OsS-WEET14 mutant alleles encode proteins without sugar transportation ability.
CR-S14 conferred strong resistance to African Xoo strain AXO1947 Zhonghua 11 contained the recessive resistance allele of OsSWEET13, which was a deletion in the PthXo2 EBE region leading to the incapability of being recognized by PthXo2 (Additional file 4). PXO86 is a Philippinesoriginated Xoo that depended on AvrXa7 to activate OsSWEET14 expression for virulence, while T7174 (NCBI: txid342109) is a Japanese strain harboring both AvrXa7 and PthXo2 for virulence. Consistent with OsS-WEET14 knockout mutant in the Kitaake background reported previously, CR-S14 conferred strong resistance to both PXO86 and T7174 (Additional file 5 and Table 1) [18]. In addition, CR-S14 showed a broad resistance to the tested Asian Xoo strains (Table 1).
To test the disease response of CR-S14 to Xoo strains that depended on TalC for virulence, an Africaoriginated Xoo strain (AXO1947) harboring TalC was inoculated on CR-S14 and Zhonghua 11 by the leaf clipping method. At least three plants of each mutant allele were inoculated with AXO1947. Fourteen days after inoculation, Zhonghua 11 showed a susceptible response to AXO1947, while CR-S14 conferred strong resistance to AXO1947 with an average lesion length less than 2 cm (Fig. 2). This demonstrated that the disruption of OsSWEET14 in the Zhonghua 11 background conferred strong resistance to AXO1947.
To determine whether the mutant alleles were still inducible, PXO86 and sterile H 2 O were inoculated on CR-S14 and Zhonghua 11 with syringes. The expression level of OsSWEET14 and the mutant alleles were measured at 0 and 48 h after inoculation using qRT-PCR. PXO86 was able to induce the expression of both mutant alleles in CR-S14 and OsSWEET14 in Zhonghua 11 (Additional file 5). This result demonstrated the mutant alleles were still inducible by AvrXa7, and the resistance response was caused by the disruption of OsSWEET14 transporter activity.
CR-S14 had enhanced plant height and normal reproductive growth OsSWEET14 transcripts were detected in various tissues of rice plants using qRT-PCR; the highest levels were measured in the stem, followed by leaf sheaths and blades (Fig. 3a). The expression pattern of OsSWEET14 was further investigated in pOsSWEET14:GUS transgenic rice plants using a β-glucuronidase (GUS) reporter gene under the control of the OsSWEET14 promoter. Consistent with the qRT-PCR result, strong GUS activity was detected in most of the cell types in the stem and mainly in the veins of leaf sheath and blades ( Fig. 3b and  d). Consistent with the expression pattern of OsS-WEET14 in the RiceXPro database [26], GUS activity was also highly detected in the roots; however, root tips, which do not have vascular bundles, lacked GUS activity (Fig. 3g). Low GUS activity was detected in the spikelet, including the palea, lemma and anther ( Fig. 3e and f). This indicated that OsSWEET14 is mainly expressed in the vascular tissues of rice plants.
To verify the influence of OsSWEET14 disruption on rice development, CR-S14 and Zhonghua11 were grown in a paddy field in Guangdong Province in China. CR-S14 and Zhonghua 11 were germinated and transferred to the paddy field at the same time, and no growth retardation was observed (Fig. 4a). Since OsSWEET14 transcripts accumulated highly in the stem, we tested whether the knockout of OsSWEET14 affected the stem diameter and plant height at the mature stage. Statistical analysis was performed using a two-tailed Student's t test against Zhonghua 11. No significant difference (P > 0.05) in stem diameter was detected between CR-S14 and Zhonghua 11 ( Fig. 4c and Additional file 6). However, CR-S14-2, CR-S14-9-I and CR-S14-9-II were approximately 7 cm taller than Zhonghua 11, which equates to an approximate 8% increase in plant height ( Fig. 4b and Additional file 6). The stem diameter and plant height were recorded for two seasons in 2019 in Guangdong, China. This suggested that the disruption of OsSWEET14 led to an increase in plant height. Fig. 2 CR-S14 confers strong resistance to AXO1947. a Lesion lengths of CR-S14 and Zhonghua 11 inoculated with AXO1947 that harbored TalC at 14 days after inoculation (n > 15). b Phenotype of CR-S14 and Zhonghua 11 at 14 days after inoculation with AXO1947. CR-S14-2, CR-S14-6, CR-S14-9-I and CR-S14-9-II rice plants harboring different homozygous OsSWEET14 mutant alleles in the Zhonghua 11 background. Zhonghua 11, wildtype control. Six-week-old rice plants were inoculated with AXO1947. Lesion lengths on inoculated leaves were scored 14 days after inoculation. Statistical analysis was performed using a two-tailed Student's t test against Zhonghua 11 In addition, we checked whether the disruption of OsSWEET14 affected the reproductive growth of rice. At least 15 plants of each mutant allele and 30 Zhonghua 11 plants were grown in the paddy field in Guangzhou, Guangdong, China. The 1000-grain weight, seed setting rate and yield of the main panicles were assessed. The assessment was executed for two seasons in 2019. The 1000-grain weight of CR-S14-2 and CR-S14-9-II was slightly higher (P < 0.05) than that of Zhonghua 11, while CR-S14-6 and CR-S14-9-I did not differ significantly (P > 0.05) from Zhonghua 11 ( Fig. 5 and Additional file 6). This suggested that the disruption of OsSWEET14 did not affect the reproductive growth of rice plants under normal growth conditions. All these results indicated that the disruption of OsSWEET14 increased plant height without reducing yield under normal growth conditions.

Discussion
In natural germplasm reservoirs, recessive resistant alleles that harbor disrupted EBEs in the promoter region are able to confer resistance to Xoo strains depending on the corresponding TAL effectors for virulence. However, TAL effectors are able to adapt to the recessive resistance alleles under the selection pressure from the resistant varieties. Recently, many PthXo2-like, AvrXa7/ PthXo3-like, TalC-like and PthXo1-like TAL effectors have been identified in Xoo strains that are able to activate the expression of OsSWEET13, OsSWEET14 or OsS-WEET11 in different rice varieties [12,13]. For example, OsSWEET13 Kit in the Kitaake background, which is the natural EBE-mutational allele of OsSWEET13, is noninducible by PthXo2. However, two PthXo2-like TALE effectors (Tal5 LN18 and Tal7 PXO61 ) identified recently are able to activate the expression of OsSWEET13 Kit to induce susceptible response [12]. These results indicate that recessive resistance can be overcome by the emergence of novel TAL effectors, and the engineering of the promoter region of OsSWEETs genes is not sufficient to confer durable and broad-spectrum resistance to Xoo. As a result, we directly knocked out OsSWEET14 in the Zhonghua 11 background directly (CR-S14) in order to confer resistance to all the Xoo strains that depend on OsSWEET14 induction for virulence since Zhonghua 11 contains the recessive PthXo2 EBE mutant allele (Additional file 4). CR-S14 was able to confer complete resistance to strains depending on both PthXo2 and AvrXa7 for virulence, such as Xoo strain T7174 (Table 1). Furthermore, CR-S14 also conferred complete resistance to African strain AXO1947, which depends on TalC for OsSWEET14 induction. However, the disease response was inconsistent with that in the Kitaake background. OsSWEET14 knockout mutants or OsSWEET14 promoter mutants in the Kitaake background are susceptible to AXO1947 [13,18]. This inconsistence implies other susceptible targets could be induced in the Kitaake genetic background instead of Zhonghua 11. In 2018, one research paper reported that TAL effector TalB MAI1 from the African Xoo strain MAI1 was able to activate the expression of two susceptible genes (OsTFX1 and OsERF#123) in the Nipponbare background and induce a susceptible response [27]. This suggested that African Xoo strains might be able to induce other susceptible genes in the Kitaake background besides OsSWEET14 for virulence. The OsSWEET14 single knockout mutant in the Kitaake background was susceptible to AXO1947. This indicated that the genetic background of rice varieties may affect the resistance response of the OsS-WEET14 knockout mutant. This hypothesis can be verified by hybridizing CR-S14 with Kitaake and checking the resistance response of F 2 generation plants. If novel susceptible targets exist in the Kitaake background, Fig. 4 CR-S14 had enhanced plant height. a CR-S14 did not show obvious morphological defects under normal growth conditions at the mature stage. b, c Performance of CR-S14 plants in terms of height (b) and stem diameter (c) relative to Zhonghua 11 plants at the mature stage. CR-S14-2, CR-S14-9-I and CR-S14-9-II were significantly taller than Zhonghua 11 (P < 0.001). The plant height of CR-S14-6 was comparable to that of Zhonghua 11 (P > 0.05). No significant difference was detected in the stem diameter between mutant lines and Zhonghua 11 (P > 0.05). Scale bar, 10 cm. Statistical analysis was performed using a two-tailed Student's t test against Zhonghua 11, and the significance is indicated by asterisks as follows: 0.01 < P < 0.05 (*), 0.001 < P < 0.01 (**), P < 0.001 (***). n > 15 F 2 generation plants harboring the homozygous CR-S14 allele will show disease-response phenotype segregation. Another possibility causing this disease response difference is the off-target effect. Mutagenesis of OsSWEET14 in the Zhonghua 11 background was mediated by CRISPR/Cas9 targeting of two target sites, Target I was in the 1st exon, and Target II was in the 3rd exon (Fig. 1). Mutagenesis of OsSWEET14 in the Kitaake background was mediated by only one target site that is the same site as Target I in Zhonghua 11 (Kittake target: 5′-GCATGTCTCTTCAG CATCCCTGG-3′ vs. Target I (CR-S14): 5′-CATGTC TCTTCAGCATCCCTGG-3′). This implies that the offtarget effect of Target II in Zhonghua 11 might lead to a difference in the disease response. This can be verified by generating a new OsSWEET14 knockout mutant in Zhonghua 11 background with CRISPR/Cas9 targeting to Target I or other different target sites.
TalC and Tal5 are two TAL effectors that target different EBEs in the promoter region of OsSWEET14. All the sequenced African Xoo strains habor TalC or both TalC and Tal5, which indicates that all the African Xoo strains are able to activate OsSWEET14 [13]. The OsSWEET14 knockout mutant in the Kitaake background is susceptible to Africa-originated Xoo strains, while CR-S14 is able to confer strong resistance to AXO1947. This implies CR-S14 may serve as a better tester line than sweet14 singleknockout mutant in the Kitaake background for the diagnostic kit for rice blight resistance [18].
CR-S14 plants were taller than Zhonghua 11 and had no reproductive defects under normal growth condition. Since OsSWEET14 had the highest expression level in the stem, the enhanced plant height might be due to the lower efficiency of sugar transportation in the stem, which needs to be studied further. In addition, Fig. 5 CR-S14 had normal reproductive growth in the paddy field. a A comparison of the main panicles at the mature stage. No obvious defects were observed under normal growth conditions. b-d 1000-grain weight (b), seed setting rate (c) and yield (d) of the CR-S14 main panicle relative to that of Zhonghua 11 at the mature stage. The 1000-grain weight of CR-S14-2 and CR-S14-9-II was slightly higher than that of Zhonghua 11 (0.01 < P < 0.05). CR-S14-6 and CR-S14-9-I had a similar 1000-grain weight to that of Zhonghua 11. No significant difference between CR-S14 and Zhonghua 11 was detected in the seed setting rate and yield. Scale bar, 1 cm. Statistical analysis was performed using a two-tailed Student's t test against Zhonghua 11, and the significance is indicated by asterisks as follows: 0.01 < P < 0.05 (*), 0.001 < P < 0.01 (**), P < 0.001 (***). n > 15 OsSWEET14 may not be responsible for reproductive development since its expression in the anther, palea, lemma and pistil was relatively low. These results imply the enhancement of plant height needs to be taken into consideration if knockout mutants of OsSWEET14 are used to confer resistance to bacterial blight. The sweet14 single-knockout mutant in the Kitaake background also showed normal growth as reported [18]. However, the homozygous OsSWEET14 T-DNA insertion mutant showed dramatic developmental defects [9]. This might be due to multiple T-DNA insertions in the rice genome, which were not discussed in paper [9].
Through comparison with the 3,010 rice genomes from 3,000 Rice Genome Project [28], we found most SNPs or indels in the OsSWEET14 genome region were in untranslated regions or introns. Two and four SNPs were identified in the 3rd and 5th exons, respectively, and 23 different indels were found in the 5th exon. However, all the indels are in the C terminal of OsS-WEET14 and do not affect the transmembrane helices. Therefore, whether those indels abrogate the function of OsSWEET14 needs to be tested further. In addition, the natural OsSWEET14 non-functional alleles need to be discovered.

Conclusions
This study demonstrated that the disruption of OsS-WEET14 in the Zhonghua 11 background is able to confer strong resistance to African Xoo strain AXO1947 and Asian strain PXO86 with enhanced plant height and no yield penalty. These results imply that CR-S14 might be a better tester line than sweet14 single-knockout mutant in the Kitaake background for diagnostic kit for rice blight resistance. The different disease responses between CR-S14 and the ossweet14 single knockout mutant in the Kitaake background might be due to the genetic background or off-target effect. This hypothesis needs to be experimentally confirmed. Moreover, natural nonfunctional OsSWEET14 mutant alleles need to be identified for further rice breeding applications. Finally, the enhancement of plant height needs to be taken into consideration when utilizing OsSWEET14 for resistant rice breeding.

Rice growth conditions
Zhonghua 11 (Oryza sativa L. ssp. Japonica cv. Zhonghua 11) was used in this study. CR-S14 is a genetically modified rice plants in the Zhonghua 11 background. At least 15 plants of different CR-S14 lines and more than 30 Zhonghua 11 plants were grown in a field under optimum growing conditions. We grew both CR-S14 and Zhonghua 11 for two seasons in 2019; the first season was from March to June, and the second season was from August to November. The paddy field is located in Guangzhou, Guangdong, China.

Bacteria strains and inoculation
All Xoo strains were cultivated on PSA medium (10 g/l peptone, 10 g/l sucrose, 1 g/l glutamic acid, 16 g/l agar, and pH 7.0) at 28°C for 2 days. Bacteria were suspended in sterile water to an OD 600 0.5 for inoculation on rice leaves. Bacterial blight inoculation was executed with the leaf-clipping method as previously described [29]. Inoculated rice leaves were collected 48 h after inoculation for gene induction analysis. Bacterial lesions were measured 14 days after inoculation. The sources of the Xoo strains tested in this study are listed in Additional file 7.

Gene expression analysis
The total RNA of rice leaves was extracted using an Eastep® Super Total RNA Extraction Kit (Promega) and reverse-transcribed into single-stranded cDNA using GoScript™ Reverse Transcription Mix, Oligo (dT) (Promega, USA). Real-time quantitative RT-PCR was executed on LightCycler480 (Roche, Switzerland) using iTaq Universal SYBR Green Supermix (Bio-rad, USA). The expression of rice ubiquitin gene 5 (Ubi5) was used as the internal reference gene. The specific primer pair for Ubi5 was 5′-AACCACTTCGACCGCCACT-3′ and 5′-GTTCGATTTCCTCCTCCTTCC-3′. All the primers used in this study are listed in Additional file 8.

Construct and rice transformation
Two target sites were selected on the 1st and 3rd exon of OsSWEET14 and were inserted into the CRISPR/Cas9 vector as described [30]. A 2-kb promoter fragment upstream of the OsSWEET14 transcriptional initiation site was amplified and inserted in front of the GUS reporter gene in the pCAMBIA1301 vector. The plasmid was transformed into Agrobacterium EHA105. Rice transformation of Zhonghua 11 was performed as previously described [31].

Determination of mutations in CR-S14
The regions flanking the two targets on OsSWEE14 in independent T 0 generation hygromycin-resistant CR-S14 lines were amplified with two pairs of primers, Target-TalC-F & R and Target-S14E3-F & R, and were sequenced to determine the potential alterations. For those rice lines with biallelic mutations, modifications were determined in segregating progeny (T 1 ) of self-pollinated T 0 plants. All the primers used in this study are listed in Additional file 8.

Determination of CR-S14 morphology
To determine the morphology of CR-S14, at least 15 plants of each line were measured. The upper diameter


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

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Genetic diversity of avocado from the southern highlands of Tanzania as revealed by microsatellite markers

Background Avocado is an important cash crop in Tanzania, however its genetic diversity is not thoroughly investigated. This study was undertaken to explore the genetic diversity of avocado in the southern highlands using microsatellite markers. A total of 226 local avocado trees originating from seeds were sampled in eight districts of the Mbeya, Njombe and Songwe regions. Each district was considered as a population. The diversity at 10 microsatellite loci was investigated. Results A total of 167 alleles were detected across the 10 loci with an average of 16.7 ± 1.3 alleles per locus. The average expected and observed heterozygosity were 0.84 ± 0.02 and 0.65 ± 0.04, respectively. All but two loci showed a significant deviation from the Hardy-Weinberg principle. Analysis of molecular variance showed that about 6% of the variation was partitioned among the eight geographic populations. Population FST pairwise comparisons revealed lack of genetic differentiation for the seven of 28 population pairs tested. The principal components analysis (PCA) and hierarchical cluster analysis showed a mixing of avocado trees from different districts. The model-based STRUCTURE subdivided the trees samples into four major genetic clusters. Conclusion High diversity detected in the analysed avocado germplasm implies that this germplasm is a potentially valuable source of variable alleles that might be harnessed for genetic improvement of this crop in Tanzania. The mixing of avocado trees from different districts observed in the PCA and dendrogram points to strong gene flow among the avocado populations, which led to population admixture revealed in the STRUCTURE analysis. However, there is still significant differentiation among the tree populations from different districts that can be utilized in the avocado breeding program.

hybridisation can occur between trees of different races when grown near each other [6,7].
Microsatellites are DNA sequences with 2 to 10 base pair repeat motifs, typically repeated 5 to 50 times [8,9]. They are spread throughout the genome, especially in the euchromatic regions of eukaryotic chromosomes, both in the coding and non-coding DNA regions [10,11]. Microsatellite regions have a higher mutation rate than other genomic regions leading to a high genetic diversity [12]. Microsatellites are also referred to as simple sequence repeats or SSRs [13]. Polymerase strand-slippage in DNA replication or recombination errors may result in differences in the number of repeats of a given motif (SSR locus) leading to new alleles at the locus under consideration. Thus, different alleles may exist at a given SSR locus, a characteristic that makes the SSRs more informative than other molecular markers, including single nucleotide polymorphisms or SNPs [14]. Being highly nformative, codominant, multi-allelic, highly reproducible and transferable among related species, SSR genetic markers have been widely used for estimating gene flow, diversity, crossing over rates and evolution for uncovering intraspecific genetic relatedness [13][14][15][16]. They have also been used in linkage map construction, for quantitative trait loci (QTL) mapping and marker assisted selection, for DNA fingerprinting of cultivars and for estimation of the degree of kinship between genotypes [17,18].
Genetic diversity refers to differences in genomic regions among individuals, populations and species. It allows populations to adapt to environmental changes. The wider the genetic diversity, the higher the chance of individuals harbouring allele variants that can help cope with given environmental changes. Such individuals will survive and transfer the favourable alleles to their offspring [19].
In Tanzania, avocado is one of the most important commercial fruits sold on domestic and international markets [20]. The first report of avocado cultivation in Tanzania dates back to 1892 [21,22]. The crop has been grown, mainly seed propagated, for over 100 years and has adapted to a wide range of topography, habitats and climates. As a result, a large diversity has accumulated in this germplasm. So far, only a single study has been conducted to assess the diversity of this germplasm using morphological traits [23]. The aim of the present study was to uncover the genetic diversity of this germplasm using microsatellite markers. The results from the study can be used to establish proper management and conservation strategies and for future breeding of the crop.

Microsatellite polymorphism and diversity
A total of 167 different alleles were recorded for the 10 loci across the 226 sampled avocado trees. The mean number of alleles/locus for all loci was 16.70 ± 1.30 (Table 1). All markers were polymorphic and detected at least 10 alleles each. The highest number of alleles per locus was 23 (AVAG22) followed by 20 (LMAV02 and LMAV29). The lowest number of alleles was 10, which was detected for locus LMAV35. The effective number of alleles ranged from 3.84 (AVAG05) to 9.59 (AVAG22) with an average of 6.81 ± 0.66. The Shannon's information index (I) ranged from 1.69 (LMAV35) to 2.59 (AVAG22). The minimum and maximum observed heterozygosity was 0.46 (LMAV14) and 0.82 (LMAV31), with an average of 0.65 ± 0.04. The average polymorphism information content was 0.82 ± 0.02 and it spanned from 0.70 (AVAG05) to 0.89 (AVAG22). With the exception of LMAV29 and LMAV31, the loci showed a significant deviation from the Hardy-Weinberg equilibrium.

Genetic diversity among the eight avocado geographic populations
Analysis of genetic diversity of the 226 avocado trees at the intra-population level revealed that for the average observed number of alleles (Na), the Njombe urban population recorded lowest value i.e., 4.20 ± 0.36, whereas both Mbeya rural and Njombe rural populations recorded peak value, i.e., 10.70 ± 2.26 and 10.70 ± 0.70, respectively ( Table 2). The mean value of effective number of alleles (Ne) was lowest (2.96 ± 0.29) in the Njombe urban and highest (5.93 ± 0.57) in the Mbozi population. For the Shannon's information index (I), the Njombe rural population had the highest average value, 1.96 ± 0.09, whereas the Njombe urban had the lowest value, 1.19 ± 0.09. The Wanging'ombe and Njombe urban populations had the lowest average values for the observed (Ho) and the expected (He) heterozygosity, i.e., 0.51 ± 0.06 and 0.71 ± 0.04, respectively. The highest value for Ho was reported in Mbozi, i.e., 0.71 ± 0.10, whereas for He, the highest value was reported in Mbozi and Njombe rural, i.e., 0.83 ± 0.03 and 0.83 ± 0.02, respectively. The lowest and highest average gene diversity was detected in Njombe urban (0.47 ± 0.09) and Mbeya rural (0.65 ± 0.11).

Molecular variance and population divergence
Analysis of molecular variance was employed to detect genetic divergence within and among the eight avocado populations. The analysis partitioned 6.08% of the variation among the populations, 17.04% among individuals within populations, and 76.87% within all individuals ( Table 3). The detected variations were significant at P < 0.0001. The total population differentiation due to genetic structure, i.e., the fixation index (F ST ), was 0.061 (P < 0.0001; Table 3). When the populations were further grouped into regions, 1.98 and 4.71% variation was noticed among groups (regions) and among populations (districts) within groups, respectively. However, the variation among individuals within populations (districts) and within individuals was similar to the values in the analysis performed on the eight populations. AMOVA conducted by grouping the genotypes into four groups according to their altitude of growth revealed that 2.52% of the total variation differentiated the groups (P < 0.0001).
The genetic divergence between the eight avocado populations was established by calculating pairwise F ST comparisons ( Table 4). 21 of the 28 pairs of populations showed a significant differentiation (P ≤ 0.05), with their F ST values ranging from 0.0111 (Mbeya city and Mbeya rural) to 0.1475 (Rungwe and Mbozi). The second highest F ST value (0.1369, P < 0.05) was recorded for Njombe urban and Mbozi.

Principal components analysis and hierarchical cluster analysis
Principal components analysis (PCA) was used to study the genetic relationships among the 226 avocado trees (Fig. 1). While the first two axes of the PCA accounted for 8.18% of all variation, most of the trees grouped irrespective of geographic origin. The sampled trees from Njombe urban tend to group to the right and so do those from Busokelo and Rungwe. The trees from the remaining populations are quite scattered in the plot. Grouping of samples from different districts or regions in the PCA plot points to genetic admixture among the sampled trees.
The genetic distance matrix of the 226 avocado tree samples was used to study the genetic relationships among the eight populations through hierarchical  clustering. The UPGMA-based dendrogram produced three major groups, each containing samples from different districts and regions ( Fig. 2), pointing at genetic admixture between samples from different districts.
Population structure and genetic relationship of the studied avocado samples Estimation of K-values, based on the methods by Puechmaille [29], revealed that the most probable K-value for our genetic data set was four (MedMeaK, MaxMeaK, MedMedK, MaxMedK = 4; Fig. 3a). This proposes that the 226 avocado tree samples can be clustered into 4 subpopulations or clusters (Fig. 3b). The genetic structure suggests a high similarity between the Busokelo and Njombe urban avocado populations as well as between the Wangingo'mbe and Njombe rural populations.
Members of Mbozi population showed highly similar genetic constitution with some members of populations from other districts, such as Mbeya city and Mbeya rural across the 10 SSR loci.

Discussion
In the present study, a total of 167 alleles were detected using 10 SSR loci across 226 sampled avocado trees with the number of alleles ranging from 10 to 23 per locus (   German and Viruel [25] and Abraham and Takrama [32] reported lower numbers, 11.4 and 11.5, respectively. Similarly, Liu et al. [33] reported the lowest number of alleles per locus, 3.10, across 10 SSR loci for 56 avocado trees investigated in Hainan Province, China. The differences between our results and the previously reported results could be due to variation in levels of polymorphism of the markers used, sample size, the diversity of the germplasm investigated and the platforms employed for resolution of amplified products [34]. The quality of genomic DNA used in PCR amplification, optimization of PCR protocols and differences in allele scoring accuracy could also be accounted for these differences. The 16.7 alleles per locus detected in the present study is comparable to that reported from other cross-pollinated species like maize (21.7 alleles [35]) and bur oak (14.3 alleles [36]). The average observed heterozygosity for the 10 SSR loci obtained in the present study was 0.65 (Table 1) which is similar to 0.64 and 0.61 obtained by Schnell et al. [30] and Guzmán et al. [31], respectively, indicating similar levels of genetic diversity for their analysed samples. Lower observed heterozygosity have been reported by Abraham and Takrama [32]: 0.48, Boza et al. [37]: 0.56, and Liu et al. [33]: 0.39, thereby pointing to a lower genetic diversity in the germplasm used or differences in the polymorphism levels of the SSR loci used by them vis-à-vis ours.
Expected and observed heterozygosity, Shannon's information index and average gene diversity are indicators of the extent of genetic diversity in populations. The analysis of diversity at the intra-population level revealed that while the observed heterozygosity, the Shannon's information index and average gene diversity were highest for the Mbozi, Njombe rural and Mbeya rural populations, respectively, the excepted heterozygosity was highest for both Mbozi and Njombe rural ( Table 2). This suggests that the Mbozi, Njombe rural and Mbeya rural populations are more diversified than the other populations and thus may offer elite materials for breeding programmes [38]. The three populations may also be able to cope with changes in environmental conditions in a better way than the other populations [19]. All the four diversity measures, except observed heterozygosity, were lowest in the Njombe urban population pointing to lower genetic diversity. This result might be attributed to the massive replacement of local seed propagated avocado with the commercial cultivars leading to a decreasing variation within the genepool. The lowest observed heterozygosity detected in Wanging'ombe, relative to the Njombe urban population, could possibly be attributed by the presence of null alleles and Most of the loci in the present study showed significance deviation from the Hardy-Weinberg equilibrium ( Table 1). Geographical structure and inbreeding within subpopulations may lead to Hardy-Weinberg equilibrium deviations. Wright's fixation indices (F IT , F ST , and F IS ) can be used to assess these within-and amongpopulation components of genetic variation. F IT measures the excess (F IT > 0; heterozygosity deficit) or deficit (F IT < 0: heterozygosity excess) of homozygotes at the global level [39]. The global heterozygosity deficit (F IT ), when AMOVA was calculated without considering regions, was 0.231 (P < 0.0001; Table 3). This implies that the observed homozygotes exceeded the expected value by about 23%. In other words, there was a reduction by 23% of observed heterozygotes relative to the expected ones. The result may suggest that avocado has a significant level of self-pollination although it is generally regarded as an out-crossing species. The SSR loci showing significant deviation from HWE could be in linkage disequilibrium with genic loci under selection in the form of heterozygote disadvantages. The fixation index, F IS , is an inbreeding coefficient that measures the excess (F IS > 0; heterozygosity deficit) or deficit (F IS < 0: heterozygosity excess) of homozygotes within a subpopulation [39]. The average inbreeding coefficient of individuals within subpopulations (districts) (F IS ) was 0.181 (P < 0.0001), which indicates that within subpopulations, the observed homozygotes exceeded the expected value by about 18%. In other words, there was a reduction by 18% of observed heterozygotes relative to the expected ones within subpopulations. At the global level, this implies that about 78% (i.e., FIS*100%/FIT) of the global heterozygosity deficit was due to the within population deficit. F ST is the fixation index that measures differentiation between subpopulations and range from 0 to 1. A value of 0 indicates that the populations under consideration are interbreeding freely (complete panmixis), while a value close to 0 indicates an unstructured population  [40]. A value of 1 infers that all genetic variation is explained by the population structure, and that the populations under consideration do not share any genetic diversity [40]. In the present study, the global degree of genetic differentiation (F ST ) for the eight avocado geographical populations was 0.061 (P < 0.0001; Table 3). This indicates that there is a significant district-based subdivision of Tanzanian avocados. This is possibly the results of mutations or genetic drift and indirect selection pressure that normally lead to lose of certain alleles or change in allele frequencies. As shown through analysis of molecular variance (AMOVA; Table 3) about 94% of the genetic variation was shared by the eight populations. The F ST value detected in our study was lower than the 0.19, 0.22 and 0.25 previously reported by Boza et al. [37], Guzmán et al. [31] and Gross-German and Viruel [25], respectively. This could be due to the fact that our study was based on samples collected from only local avocados (excluding commercial cultivars) and sampling involved only three nearby geographic regions in Tanzania. Contrary to our study, Boza et al. [37] studied avocado samples from United States and Mexico representing Persea americana (218 samples), P. nubigena (2 samples) and P. kruguii (1 sample). On the other hand, Guzmán et al. [31] analysed only P. americana collected in one country comprising local selections, root stocks and commercial cultivars. Similarly, the 315 samples analysed by Gross-German and Viruel [25] included also 5 samples from P. longipes, P. nubigens and P. schiedeana. However, the average F ST in the present study is Fig. 3 Population structure of the analysed trees. a: Identification of the optimum K-value using four different approaches developed by Puechmaille [29]; b: Population structure of 226 avocado trees suggesting varying levels of genetic admixture from four origins (colours) in the eight districts in the southern highlands of Tanzania comparable to the 0.05 reported by Cañas-Gutiérrez et al. [15] for 197 avocado samples from Colombia.
Analysis of molecular variance showed that 6.08% of the total genetic variation was partitioned among the 8 districts (Table 3). When the samples were grouped according to elevation ranges, the genetic variation among groups was 2.52%. These results concur with that of Teshome et al. [41], who observed lower differentiation among altitude-based groups of Ethiopian field pea (Pisum sativam L.) populations (2%) compared to that of region-based groups (8%).
In the present study, principal components analysis (PCA) and hierarchical cluster analysis were used to study the genetic relationships among the sampled trees. Neither the PCA nor the dendrogram separated these trees according to their districts or regions. This was in line with AMOVA findings ( Table 3) which showed that about 94% of the total genetic variation was shared by the populations. The PCA and dendrogram findings are also supported by the population pairwise F ST that revealed absence of differentiation between pairs of populations, such as Rungwe versus Njombe urban and Buokelo versus Njombe urban. Similar results were obtained when these avocado trees were characterised with morphological markers [23]. Genetic admixture among the avocado populations is attributed to sharing of seeds between farmers from different districts and selling of avocado produce from one district to another where the seeds could then be planted. It may also be due to introduction of highly similar germplasm to more than one districts/regions.
The model-based STRUCTURE was used to study the population structure of the 226 avocado plants. The results showed that the sampled plants can be regrouped into four clusters based on their genetic characteristics detected at the 10 studied loci. High similarity in population structure was noticed between the Busokelo and Njombe urban avocados as well as the Wangingo'mbe and Njombe rural avocados. This result is supported by lack of differentiation among these pairs as shown by the population pairwise F ST values (Table 4). Each geographic population (district) have alleles originated in at least three STRUCTURE based populations (clusters). Various analyses such as AMOVA, global F ST , PCA and UPGMA revealed that avocados grown in different districts of Tanzania show high genetic similarity with low but significant genetic differentiation between them.

Conclusion
High diversity was detected in the analysed avocado germplasm based on standard and molecular diversity indices. These findings implies that this germplasm is a potentially valuable source of variable alleles that might be harnessed for genetic improvement of this crop in Tanzania. The principal components analysis and hierarchical cluster analysis showed a mixing of avocado trees from different districts, pointing to strong gene flow among the eight populations. This is in line with the results of the model-based population structure analysis that revealed that the alleles of each district based populations originated from at least three of the four genetic populations.

Collecting samples and DNA extraction
Samples were collected in three regions; i.e., Mbeya, Njombe and Songwe, which are located in the southern highlands of Tanzania. From these regions a total of 41 villages or streets were visited across eight districts that are renowned for harbouring many seed propagated avocado trees. Locations of the study sites are given in Fig. 4. The number of trees sampled in each district and region is presented in Table 5 whereas some data on climate of the studied districts are presented on Table 6.
We visited the study sites from February to August in 2017 and young, leaf samples were collected from a total of 226 seed-propagated avocado trees. We sampled four to six leaves from each tree and then packed them in porous tea bags (two to three leaves per bag). The bags were then put into plastic bags followed by addition of silica gel to dry the leaves. When needed, we replaced the worn-out silica gel and continued doing so until the leaf samples were completely dry. Complete dryness was determined as when added silica gel retained its original colour. We extracted DNA from the dried young avocado leaf samples using the Thermo Scientific Genomic DNA Purification Kit following the manufacturer's instructions with minor modifications. DNA integrity was checked in a 1.2% agarose gel electrophoresis and its quality and quantity was assessed with a NanoDrop Spectrophotometer.

Microsatellite analysis
Forty microsatellite markers were selected among those developed by Sharon et al. [24] and Gross-German and Viruel [25] based on their reported levels of polymorphism. The primers were tested on eight avocado samples from eight populations, each representing a different district. Thereafter, 16 microsatellite markers that were highly polymorphic within the eight samples were chosen and used for analysis of all samples. However, only 10 of the 16 markers showed consistent amplification and the data analysis is therefore based on these 10 markers. Background information about the microsatellites, such as names and repeat motifs, is presented in Table 2. Amplification of target microsatellite loci was carried out in a total reaction volume of 25 μl containing 2.5 μl of 10X PCR buffer, 1.5 μl of 25 mM MgCl 2 , 0.3 μl of 25 mM dNTPs, 0.75 μl of 10 μM of each forward and reverse primer, 0.2 μl of 5 U/μl Taq polymerase and 25 ng genomic DNA. PCR reactions were run in a S1000™ thermal cycler (BIO RAD, Hercules, CA, USA) using a program of initial denaturation at 94°C for 1 min, 35 cycles of 1 min denaturation at 94°C, 30 s annealing depending on the specific primers' annealing temperature and 1 min extension at 72°C, followed by a 10 min final extension at 72°C. The size of the PCR products were determined using an Applied Biosystems 3500 Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA, USA).

Data analysis
Standard diversity indices for markers; i.e., observed and effective number of alleles, observed Nei's [27] and expected heterozygosity and Shannon's information index [28] were computed using Popgene32 software version 1.32 [48]. Polymorphism information content for each SSR locus was assessed using Cervus version 3.0.7 [49]. Hardy-Weinberg equilibrium tests (molecular diversity index) were carried out in Popgene32 software version 1.32 [48]. To assess diversity at intra-population level, we computed standard diversity indices for each geographical population (district) using Arlequin 3.5.2.2 [50]. The indices calculated were observed number of allele and observed and expected heterozygosity. The effective number of alleles and Shannon's information  index were computed using GenAlEx version 6.5 [51].
We also estimated average gene diversity across all loci for each population in Arlequin 3.5.2.2. Analysis of molecular variance (AMOVA) was carried out in Arlequin 3.5.2.2 under 1000 permutations, 100,000 steps in Markov chain and 10,000 Dememorisation Steps. Hierarchical global AMOVA was conducted by grouping individual samples according to districts, regions and elevation ranges. Arlequin 3.5.2.2 was used to compute fixation indices (F ST , F IT , F IS, F CT and F SC ) and pairwise comparisons between the eight geographical populations.
We employed principal components analysis (PCA) to display the genetic relationships among the eight avocado geographical populations (districts). Allele composition for each tree was computed in the adegenet R package [52] and then used in PCA in XLSTAT version 2019.4.2 [53]. Hierarchical cluster analysis was also deployed to assemble avocado samples with similar genetic characteristics across the 10 loci in the same group. In order to achieve this, we computed Nei's genetic distance in GenAlEx and imported it in MEGAX [54] where the dendrogram in the newick format was produced using the unweighted pair group method with arithmetic mean (UPGMA) [55]. The dendrogram was visualized and customized in the Interactive tree of life (iTOL) v4 [56]. To identify genetic populations for the 226 avocado trees, we performed a Bayesian cluster analysis on the allele dataset using STRUCTURE 2.3.4 [57][58][59]. The admixture model was adopted with 10,000 burn-in period and 100,000 Monte Carlo Markov chain iterations. The range of K, the probable number of subpopulations or clusters, tested was 1 to 10. For each K value, we performed 20 independent runs. The structure results were analysed and visualised in STRUCTURE SELECTOR [60] where the optimal number of genetic clusters was computed based on distinct approaches; the median of medians (MedMedK), median means (Med-MeaK), maximum of medians (MaxMedK) and maximum of means (MaxMeaK) [29].


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Photosynthetically active radiation in strawberry produced in stair-like containers

In intensive hydroponic systems, such vertical and pyramidal systems, photosynthetically active radiation (PAR) and integrated (IPAR) may be intercepted by the containers and the leaf area of the plants grown in the upper strata, decreasing fruit yields and quality. The objective of this study was to evaluate the behavior of PAR and IPAR at different planting heights and the effect of geographic orientation and fruit yield and quality of strawberry produced in stair-like containers. The factors were four heights of the container (0.20, 0.50, 0.80 and 1.10 m) and two orientations (east and west). At each height and orientation of the container, PAR and IPAR, yield and fruit total soluble solids were measured. PAR and IPAR were statistically different at all heights evaluated and were highest in the highest containers. The east and west orientation of the containers was significantly different in the morning and in the afternoon for PAR and IPAR, while daily average values were equal. Yield on average was 47% higher in plants cultivated in containers at heights of 0.50, 0.80 and 1.10 m, relative to those in 0.20 m high containers. Fruits grown in the highest containers had higher values of total soluble solids.

Received on May 30, 2019; accepted on December 13, 2019
this area is field cultivated under full sunlight with 4.62 kg m -2 average yield (SIAP, 2018), considered low. On the rest of the area some sort of technology is used, such as mulching and fertigation (PRNF, 2012). Of this production, 70% is exported to Canada, the United States, Brazil, Saudi Arabia and the United Kingdom (SAGARPA, 2016). During the last decade, demand has increased so that it has been necessary to incorporate new technologies to increase yields, such as plastic covering in protective structures, drip irrigation and hydroponics. Intensive hydroponic is not enough light, growth is slow and fruit quality decreases, and when there is excessive light, photosynthesis does not increase, nor does plant grow. In the leaf, photosynthetic efficiency is higher with low levels of radiation (Norman & Arkebauer, 1991) because the leaves become saturated with radiation when they are exposed to direct sunlight.
Worldwide, Mexico is the third producer of strawberry. In 2018, the cultivated area was 13,563 ha (SIAP, 2018). The states with the largest cultivated area are Michoacán, Baja California and Guanajuato; 69% of O ne of the functions of plant leaves is interception of the solar radiation necessary for photosynthesis and production of carbohydrates. Of the radiation the earth receives, only the ones between 0.400 and 0.700 µm are used; this is the photosynthetically active radiation (PAR) (Oyarzún et al., 2011), which affects crop growth and development by its effects on photomorphogenesis and mutamorphogenesis (Johkan et al., 2012). Each plant species has an optimal range of PAR that maximizes photosynthesis and growth. When there systems have several great advantages over the traditional system in soil and full sunlight, for example, efficient use of water and fertilizers, and optimization of crop space and management. Moreover, in small areas, vertical or horizontal hydroponic systems permit increased planting density and, consequently, yield, while guaranteeing fruit safety.
To optimize greenhouse area in protected and intensive agriculture, strawberry cultivation in suspended containers or in vertical systems has been studied (Fernandes-Junior et al., 2002). Using vertical containers increased yields up to 100%, relative to use of horizontal containers (Fernandes-Junior et al., 2002). In tall horticultural species, such as tomato (L. esculentum), pyramid and stair-like systems have also been used (Sánchez-del-Castillo et al., 2009) with which high yields have been obtained. In these systems, plants grown in the upper canopies achieved higher yields than those grown in the lower canopies (Sánchez-del-Castillo et al., 2014) because they received more PAR. In strawberries grown in vertical systems, Calvete et al. (2007) found that yield decreased from the upper to the lower part and attributed the difference to differences in PAR. The decrease in PAR was due to the shade caused by leaves of the plants in the upper part. In vertical systems the upper canopies use a part of the incident radiation; the rest passes unintercepted through the holes in the canopy and reaches the ground (López-Lozano et al., 2007) or through spaces with no leaves or stems. These spaces can be up to 40% (España-Boquera et al., 2015). This space is useful in stair-like containers with strawberries since it allows direct solar radiation to reach the lower canopies, which also make use of diffused radiation that can reach up to 30% (Higashide, 2008).
Plant species require different levels of IPAR, ranging from <5, 5-10, 10-20, 20-30 and >30 mol m -2 d -1 . Strawberry plants have requirements between 10 and 20 mol m -2 d -1 (Torres et al., 2011). IPAR also determines metabolite concentration in the plants (Gent, 2014): the more the radiation, the higher the content of sugars and dry matter (López & Runkle, 2008). The objective of this study was to evaluate the behavior of PAR, IPAR and fertigation at different heights and geographic orientation of strawberries in stair-like containers and their effect on fruit yield and quality.

Stair-like hydroponic modules
In the 20×8×5 m (length × width × height) tunnel-type greenhouse covered with 200 µm white polyethylene, three stair-like modules were constructed to cultivate strawberries in hydroponics. For each module, eight white PVC tubes, 15.24 cm diameter by 6.0 m long, were used. The final dimensions of each model were 1.4×3.0×1.10 m (width × length × height). These modules were placed in such a way that four tubes were facing east and the other four faced west. The tubes were placed 0.20, 0.50, 0.80 and 1.10 m above soil level ( Figure 1).
On the upper part of the tubes (containers) 4 cm orifices were made every 25 cm and filled with composted wood bark shredded to 3 mm; its physical properties were 74% moisture retention, 1.66 g cm -3 particle density, 0.46 g cm -3 bulk density, 72.40% total pore space, 14.30% aeration capacity and 24.20% easily available water. In each orifice, a strawberry runner (Fragaria ananassa) cv. Camino Real was planted.

Treatments
The treatments were four container heights (0.20, 0.50, 0.80 and 1.10 m) and two orientations (east and west). Each treatment was replicated three times and the experimental unit was one lot of 12 plants. An experimental design of complete random blocks was used with factorial array.

Photosynthetically Active and Integrated Radiation
PAR (µmol m -2 s -1 ) was recorded every hour from 8:00 to 16:00 h, two days a week during the entire crop cycle. PAR was measured outside the greenhouse 1.10 m above ground level and inside above the canopy of the plants in each container with a portable linear quantum radiation meter MQ-301 (Apogee Instruments ® , Inc. UT. USA). IPAR mol m -2 day -1 was obtained following Chang et al. (2008) at each height and orientation of the crop.
Fruit yield and quality Fruits were harvested from January to June 2016. Yield was obtained following norm NMX-FF-062-1987, considering the sum of the weekly fruit harvest during the entire productive stage of the crop. The content of total soluble solids (°Brix) was determined in a sub-sample of three fruits per picking, with a digital refractometer (HI 96801, HANNA Instruments ® ).

Statistical analysis
The obtained data were subjected to analysis of variance and the Tukey multiple comparison of means.
For the variable yield and IPAR, a correlation was performed with a fit to a second order polynomial regression (Montgomery, 2006). For all analyses, the statistical software SAS, version 9.0 (SAS Institute, 2002) was used.

Analysis of variance
The analysis of variance shows significant statistical difference in all evaluated variables for the factor height. These differences are due to differences in photosynthetically active radiation the plants received at different heights. For the factor orientation and combination of factors, none of the variables was statistically different. The similarity among variables may be due to the similar PAR the plants received during the day in the two orientations.

Fertigation parameters
The different container heights were significantly different (P≤0.01) in the variables pH, EC and percentage of fertigation drainage (Table 1). The highest values of pH (7.49 and 7.65) were obtained in the drainage of the containers placed 0.80 and 1.10 m high; these containers also had the highest values of EC (1.32 and 1.37 dS m -1 ). Only the 1.10 m high container had the lowest percentage of drainage (12.33%). These differences occurred because the highest container received more PAR and IPAR, which induced higher water uptake, a decrease in drainage and an increase in concentration of salts. Morales & Urrestarazu (2013) observed similar results in a crop of hydroponic tomatoes.
Temperature, relative humidity and global radiation During the crop cycle, average temperatures inside and outside the greenhouse were 27.5 and 18.5°C, while average relative humidity inside and outside the greenhouse was 45.5 and 69.7%. According to Kadir & Sidhu (2006), these values of temperature and relative humidity are suitable and favor strawberry development and production. Average monthly global radiation recorded inside the greenhouse from January to June was 11.7, 12.9, 14.0, 15.7, 16.3 and 14.1 MJ m -2 d -1 , respectively, and was within the range required by strawberries (Torres & Lopez, 2011).

P h o t o s y n t h e t i c a l l y A c t i v e Radiation
PAR was significantly different (P≤0.01) for the east and west orientation and container heights at 09:00, 12:00 and 16:00 hours (Figure 2a, 2b and 2c, respectively), but between orientations,  Photosynthetically active radiation in strawberry produced in stair-like containers The IPAR received by strawberry plants cultivated in stair-like containers was significantly different among heights and different from the exterior IPAR, which reached the maximum value (49 mol m -2 d -1 ). The highest IPAR was registered on plants in the 1.10 m high container with 29.8 mol m -2 d -1 . This value of IPAR decreased in function of lowering heights of the containers to the lowest value of 19.0 mol m -2 d -1 in plants of the container at 0.20 m high. All the plant heights had IPAR values above those reported by Bradford et al. (2010), Sonsteby & Heide (2006, Verheul et al. (2007) and Casierra-Posada et al. (2012) in greenhouse strawberries.
Strawberry plants have a low IPAR requirement (10-20 mol m -2 d -1 ), unlike other plants such as tomato (S. heights captured more PAR than the 400 µmol m -2 s -1 captured by greenhouse strawberries, cultivar Honeoye, reported by Bradford et al.(2010), and more than the 270 µmol m -2 s -1 reported by Sonsteby & Heide (2006) in the cultivars "Korona" and "Elsanta". The PAR obtained in this study at different heights and orientations of the containers was higher than the PAR required by the species, which oscilates between 200 and 400 µmol m -2 s -1 (Torres & Lopez, 2011). Therefore, with good crop management, which includes timely irrigation, fertilization, and pest and disease control, yields can be higher than that of the traditional system in soil since PAR is not a limiting factor.
Integrated Photosynthetically Active Radiation average daily PAR received was not different. However, it was different from the PAR outside the greenhouse ( Figure  2d), whose maximum was 1300 µmol m -2 s -1 , much higher than the 900 µmol m -2 s -1 obtained at midday inside the greenhouse and at the highest containers. This behavior of PAR is due to a higher angle of incidence of the sunrays both in the morning (east orientation) and in the afternoon (west orientation) and the vertical rays at midday. The strawberry plants cultivated in the containers 1.10 m high received an average of 842 µmol m -2 s -1 during the day, while those that grew 0.20 m above the ground received 502 µmol m -2 s -1 ; that is, the higher plants received 67% more PAR than the lower ones.
Plants growing in containters at all lycopersicum), which needs 30-35 mol m -2 d -1 (Hernández & Kubota, 2014). Therefore, the IPAR values obtained at different plant heights on the stair-like system satisfy the requirements for this species.

Yield
Total strawberry crop yield in the stair-like system had significant differences (P≤0.05) among plant heights (Figure 3). Yield of plants grown in containers placed 0.50, 0.80 and 1.10 m high were not significantly different and were on average 47% higher than yield of plants cultivated in 0.20 m high containers. The low yield of strawberry plants in the lowest container was associated with a decrease in PAR caused by shading by the upper containers at some point in the day, coinciding with results with tomato (Solanum lycopersicon) reported by Sánchez-del-Castillo et al. (2014), who indicate that in a stair-like system the lower containers receive less radiation and produce fewer floral buds and, thus, yield is lower.

Total Soluble Solids
In the months of lower temperature and radiation (January and February) there were no significant differences in total soluble solids in fruit among container heights. However, differences were found in the three months of harvest (Table 2). This is likely due to the increase in PAR and temperature. The higher content of total soluble solids was obtained in fruits produced in the containers higher than 0.50 m, achieving values of 9.26, 9.92 and 11.45ºBrix, respectively. This indicates that higher  IPAR increases the content of soluble solids in strawberries. Ayub et al. (2016) reported values of total soluble solids in the range of 6.57 and 7.93ºBrix in the cultivar Camino Real, similar to those obtained in the lowest container during the entire crop cycle, while Cecatto et al. (2013), with the same cultivar, obtained 5.43ºBrix, which is lower than that obtained in fruits produced in the 0.20 m high container. Casierra-Posada et al. (2011) concluded that diffused and direct radiation directly affect the sugar content in fruits. However, our study shows that the content of total soluble solids in strawberry fruits produced in the stair-like system in a greenhouse did not decrease considerably.

C o r r e l a t i o n b e t w e e n Photosynthetically Active Radiation and Yield
Yield of strawberry cultivated in containers 0.50, 0.80 and 1.10 m high correlated significantly (P≤0.05) with IPAR, with high R 2 : 0.88, 0.96 and 0.81 (Figure 4b, 4c and 4d), respectively. Correlation between yield of plants grown in 0.20 m high containers and IPAR was low, with an R 2 = 0.40 ( Figure  4a). These results are similar to those obtained by Kjaer et al. (2012), who obtained a linear relationship between accumulated dry matter and PAR, indicating that fresh matter increases proportionally with dry matter when PAR increases. The high values of R 2 of the three higher containers, relative to that of the lowest container, confirm the positive relationship between PAR and higher yield of strawberries grown in stair-like systems.
It is concluded that the Incidence of PAR and IPAR received by strawberry plants at all heights of the containers in the morning was significantly different from that received in the afternoon in function of geographic orientation. However, the daily average values were similar. Yield was significantly higher in containers at 0.50, 0.80 and 1.10 m high than in the 0.20 m high container, 47% higher. The higher the container, the higher the content of total soluble solids in strawberry fruits during the months of higher temperature and radiation. The strawberry production system of stair-like containers is a viable option for increasing strawberry yield and quality in intensive protected systems.


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Microbiological Assessments in a Cut Flower Crop Polytunnel Field Trial Adopting Soil Covering and Microbiocides for Fusarium Wilt Suppression

Cut flower Matthiola incana were raised by local commercial cultivators in a polytunnel. The field soil beds were either left uncovered as normal or covered with polythene sheets (except a hole for plant plug space). Average temperatures in the top 5 cm soil under cover dropped from 28 C to 18 C compared to its spiking up to 37 C in uncovered counterparts. Microbiological analyses indicated that soil covering induced two log10 folds reduction of the wilt causal fungi Fusarium oxysporum and concomitantly increased one log10 fold wilt antagonistic natural soil inhabiting fungi populations. Standard dip/drench mixtures of commercial and local isolates microbiocides (bacteria) applied to M. incana plug roots improved plant health assessed by visible scores of the level of damage or wilt symptoms under soil covered treatments. Scanning electron microscopy, cultural and 16S rRNA PCR analyses revealed potent antifungal bacteria attached to the hyphal surfaces of F. oxysporum as ectosymbionts that may have implications for virulence regulation and host plants’ wilt disease control. Our microbiological data support the prospects of combining physiological and microbiological interventions upon covering the soil surface that offers the local horticulturists an evidence based sustainable means of Fusarium wilt control suppression in polytunnel crops.

I. INTRODUCTION
Fusarium wilt of ornamental plants such as Brompton Stock (M. incana), Carnation (Dianthus) and Narcissus important in the cut-flower market in Northern Ireland and is the most frequently encountered glasshouse, polytunnel (polythene covered tunnel) raised crop disease, followed by other soil-borne phytopathogens Pythium and Rhizoctania [1] on the basis of their host specificity causing crown, root rots and vascular wilts. Fusarium wilt disease control measures pose a formidable challenge to the glasshouse crops industry [2] due to stringent Europe-wide pesticide usage regulations, high costs, environmental, human and soil health impact. The sustainable biological alternatives [3] for Fusarium wilt control include soil drenches, root dips of commercial microbiocides (Serenade Max, Prestop), mycoparasitic fungi Clonostachys rosea [4]. Northern Ireland cut flower growers also adopt spent compost dressings and crop rotation to mitigate the flower plants wilt, by planting wilt resistant racespecific [5] varieties lettuce (Lactuca sativa) providing extra farm revenue during off-flower [6]. Subsequently, the flower grower observed (personal communication) surprisingly, visible healthy looking cut flower crop in randomly polythene sheet covered polytunnel field soil plots.
Normally soil coverings in an open filed render rise in subsoil temperature, promote seed germination, prevent avian menace and increase surface saprophytic fungal populations. However, in a polytunnel, the covering of soil in local flower crops suggests light, temperature lowering environmental effects usually associated with circadian clock signal modifications that tend to regulate fungal development, in particular those of F. oxysporum [7] in the critical plant stem base/soil microbiome zones [8], [9]. However, despite the above valuable literature background, core microbiological supportive data in the stem wilt infection zone concomitant to the covering of soils under polytunnel field conditions are scarce. In this study, prompted by the flower grower's visible observations on healthy looking plants discovered by chance in plots with soil covering, we set out to seek the microbiological premise, and investigate the physiological on-field (soil covering) manipulation, and the scientific outlook for such practicable and grower adaptable steps in enhancement of disease management of the opportunistic soil phytopathogen F. oxysporum, affecting cut flower crops, such as M. incana.

A. Polytunnel field trial
Using field soil covered by the polytunnels of one of the local cut-flower growers (

C. Microbiological investigations 1. Isolation of wilt fungi and competing antagonistic fungi from flower bed soil
The list of organisms used in this study are described in Table1. Fusarium genera wild type fungal colonies were normally distinguishable in Potato Dextrose agar (PDA) plates due to their distinctive colour and hyphal features (white-mild pink hypal extensions, intense carmine red pigmentation). Other fungi and bacteria co-occurring as adjacent colonies on the same agar plates exhibiting antagonistic zones against presumptive Fusarium colonies on PDA were carefully subcultured for further studies. Type collection cultures of Fusarium oxysporum f. sp. mathiolii (16602 and 16603) were obtained from LGC Standards, Teddington, Middlesex, UK for molecular comparisons with wild-type isolates.
2. Isolation of fungal hyphae surface resident bacteria, scanning electron microscopy and molecular assays Stem sections of putatively infected M. incana were surface sterilized and then placed on PDA. The emergent hyphae were serially subcultured a minimum of three times onto fresh PDA. These purified Fusarium WT were examined to check for any co-colonising bacterial cultures adherent to the hyphae (as the presence of a glossy slime). These bacteria were carefully isolated and single-colony purified on LB agar for further molecular analyses. The presence of bacterial ectosymbionts on hyphae of WT Fusarium was further examined via Scanning Electron Microscopy (SEM). PCR assays for the amplification of either fungal 18S rDNA ITS regions, or 16S rRNA universal bacterial amplicons were employed to establish the identity of WT Fusarium morpho-types and those of the hyphal surface bacteria respectively. The resulting PCR amplicon sequences obtained for both the fungal and bacterial samples were established from chromatogram analysis [10] and the confirmed set of sequences were compared with those stored in the GenBank using the BLASTn alignment software (http://www.blast.genome.ad.jp/).

Effect of temperature on in vitro growth vigour of Fusarium spp.
From stock Fusarium species cultures 6mm diameter plugs were excised and transferred individually to the Cartesian coordinate centre of four directional segments marked previously using a fine-tip marker pen of fresh plates of PDA, incubated for 3 days at ambient temperature to facilitate natural contours of hyphal growth to advance. Once the culture plug establishes, the plates were incubated at temperatures viz., 4 o C, 10 o -15 o C, 16 o -19 o C (ambient), 20 o -35 o C) reflecting the spectrum of Poly tunnel canopies and soil thermal dynamics known in Northern Ireland. The culture plates were examined using a binocular microscope and the outline of the perimeter of the hyphae was carefully traced by marker pen. At 3-day intervals, the extent of the hyphal growth (mm) was marked up to 3 weeks. The final area covered by each culture was measured using the bio-imaging technique [11] to assess growth vigour using the Autochemisystem UVP Bioimaging system (UVP Products, Cambridge, UK), supported by LabWorks software package. Initially the instrument was set on white light and to an exposure ratio of 490:500, with a constant focus of 47% calibration. Using the "Area Density" tool, (in pixels) the entire 'area of individual plate' was measured first, recorded, and then using freehand 'draw' tool followed on by the 'area of irregular contours' of fungal growth co-integrated within the Cartesian coordinates to give an accurate estimate of the in vitro growth intensity of Fusarium species set against the challenge of temperature regimes during their incubation.

Statistical analyses
The arbitrary ratio, fungal growth, was calculated for each treatment as the ratio of total surface area occupied by fungal growth/total surface area of the petri dish. Statistical analyses on microbiological and plant health investigations were performed employing the student t-test and values >0.05 (5%) was considered not significant. The temperature ramps recorded over a 8-week period when the soil surface was covered with the polythene sheets showed ( Fig. 1) a dramatic drop from 28 o C to 18 o C within the first week of plant growth, and after flux of ± 2 o C for the next weeks, the temperature dipped down to 14 o C for the ensuing 4 weeks before normalising at 18 o C for surface to root depth layer up to 5 cm deep. The relative sharp dip in the temperature, by a margin of over 10-14 o C during the crucial phase of M. incana host plug plant establishment, is also important for the growth and proliferation of the saprophyte wilt causal agent F. oxysporium. As such, it is a critical time window for host-pathogen interaction. On the other hand, in the uncovered soils, while the surface ambience also had initial fall in temperature from a maximum of ±37 o C down to 28 o C, the soil bed beneath had an average of 24 o C; the surface ambient temperature and the MHPRZ (5 cm deep) soil in covered state were divergent throughout the first 8-week period recorded and lacked any normalisation of the two thermal ramps compared to the synchronisation and harmony found in temperature gradient in the surface versus underground 5 cm deep covered soil. Increased symptoms of wilt in Chrysanthemum morifolium when cultivated in higher temperature (> 24 o C) in greenhouses in the USA [12] during summer seasons have been a well-known phenomenon; symptoms became more severe when temperatures ≥27 o C, such as those recorded in our studies on M. incana cut flower plantlets raised in polytunnels in Northern Ireland. The apparent divergent patterns of thermal flux between the covered and uncovered soil environment could have impacted the population dynamics of the emergent fungi colonising the plug plant host (M. incana) roots.

B. Plant health evaluations: effects of soil covering and application of microbiocides
Two cultivars CV1 and CV2 of Matthiola incana (supplied to the flower grower as plug plants by a stockist based in The Netherlands). Scores of plant health were based on the level of damage or wilt observed: 1=(no damage), 2=(10%), 3=(11-25%), 4=(26-50%), 5=(100% dead). Error bars indicate the standard deviations of the mean.
Plant health scores (Fig. 2) showed the percentage of plants compared to their score damage per treatment and cultivar (CV1, CV2) variations against covered and uncovered regimes. Plants with covered roots generally exhibited higher number of plants with less damage when the bacterial treatments were administered as a plug drench, while in uncovered plants, a wider range of symptoms were apparent on visible scores basis. Both covered and uncovered plants secured better and higher scores when a drench treatment followed a dipped mode of treatment before planting the plug plants in the pots. For example, when plants were uncovered regardless of the mode of bacterial pre-treatment (dipped or drenched) or post treatment, over 60% of CV2 plants had only 10% damage, whereas over 30% of CV1 had 10% damage. However, in the case of soil covered plants, drenched method of pre-treatment of plugs showed CV2 plants had only succumbed to 10% of wilt damage, whereas over 60% of CV1 had 10% damage. Control treatments in general which received no bacterial treatment had a higher number of plants (≥10%) with wilt damaged appearance. Overall visible scores indicated that plug plants grown under covered soil seemed to be healthier plants and showed more vigour. The covered treatment scores were generally lower (less damage to plants) therefore the differences between treatments and control (no microbiocides) was less. However, in the uncovered treatments, plants were either dead or dying (≥5) and there were more overall variations in scores (≥2). From the visual observations it can be seen the covered plants look healthier and bigger than the uncovered. Our preliminary visible scores data upon plant health ( Fig.  2) also heralds the importance of circadian influence on fungal pathogenesis which corroborates those of several researchers who have highlighted the regulation of growth, metabolism, and biotic and abiotic stress resistance responses to light upon a number of fungal pathogens including Fusarium spp [13], Aspergillus spp [14], Phytophthora spp [15]. Our results also heightens the need for utilizing natural physiological tools (e.g. covering the soils around the stem base and roots) as a practical supplementary means for enhanced suppression of the wilt pathogens in tandem with current trends of biological control measures of dipping or drenching treatments (e.g. microbiocides) of plant plugs in field conditions. The systemic toxin theory [16] considers that toxins, (such as fusaric acid produced by F. oxysporum) disturb the metabolism of the infected host plant, resulting in leaf wilt with a consequent reduction in root growth, leading to apoptosis, necrosis, and even death. However, in our experiment, our primary goal insofar as heat and light effects are concerned was to evaluate the overbearing influences that the soil covering manipulation has, on the saprophytic soil inhabiting F. oxysporum in the soil surface and Matthiola host-plant root systems (MHPRS) before it penetrates the host plant cells.

C. Effect of temperature on in vitro growth of Fusarium species
Our preliminary experiment (Fig. 3) to gauge the effect of temperature on in vitro growth vigour of Fusarium spp. in PDA plates incubated in temperature gradients viz., (4 o C, 10 o C -16 o C (ambient), 19 o C (mean annual polytunnel temperature in local flower growers yards), 25 o C-35 o C (polytunnel in this study)) indicated that local native Fusarium spp. were heat sensitive and their growth vigour significantly (P= 0.005) varied sharply, based on the temperature of their environment. In our study, we covered the soils with polythene to simulate a physiological effect of induced light, temperature as compensates of environmental (hidden) factors in air or immediate underground beneath the soil [17]. It is thus perceivable from our generic experimental results presented in this study, the 'light' perception of host (M. incana) canopy above the ground would be the same for covered or uncovered soils, However, for sessile saprophytic fungi such as Fusarium spp, it is critical for the saprophytic mycota to especially evade the substrate in the harsher temperatures prevalent in top surface soil and either head beneath in the competitive substrate-rich root rhizosphere, or produce spores for spreading in the soil/plant interface (e.g emerging stem area near top soil) environment for expressing a temperature modulated opportunistic pathogenicity, as it is apparently the case in M. incana stem wilt disease development. Thus, lowering of temperature at this critical soil-plant interface (MHPRZ) will limit the progress of fungal growth and development and besides the ensuing physiological (light, temperature modulated) molecular plant-pathogen interactions.

D. Microbiological assays
The soil samples were obtained from a local flower grower's farm (Greenisland Flowers, Co Armagh, Northern Ireland) and Fusarium spp. amongst other fungi were isolated from soil collected from polytunnel which had a history of Fusarium infestation of M. incana. The soil sampling was carried out closest to the plug plant roots and the stem base where wilt damage was most evident, when the plug-plant transplants were 3-4 weeks old. Concomitantly, a piece of infected stem tissue of M. incana which had been surface sterilised and then placed on PDA yielded emergent presumptive Fusarium hyphae from infected tissue. Soil samples taken from closest to the base of the stem and emerging Matthiola host plant root zone (MHPRS) yielded a mixed population of bacterial and fungal colonies on PDA plates (Fig. 4 a). On certain of these plates there was evidence of inhibition of fungal growth, due to bacterial antagonism of the expansion of the hyphae (data not shown]. Single purified Fusarium colonies were incubated and plates with fungal hyphae swarmed by bacterial satellite colonies were retained.

E. Scanning Electron Microscopy (SEM)
The Fusarium hyphae obtained from the infected tissue were transferred on to fresh PDA plates, following which bacterial colonies were visible surrounding the emergent mycelial hyphae. Scanning electron microscopy (SEM) of the fungal hyphae from these culture plates revealed the presence of ectosymbiotic rod-shaped bacteria (Fig. 4 b, c) attached to the wilt pathogen Fusarium oxysporum hyphae (obtained via the wilt disease prone Mathiola incana infected tissue). Interestingly Paenibacillus polymyxa, was amongst potent antifungal native bacteria isolated from our previous study alongside Bacillus subtilis and B. amyloliquefaciens; all three of which were demonstrated in vitro [18] for their efficacy to act as natural alternative microbial source for wilt control in floriculture. The generic physiological mechanisms of the pathological wilting of higher plants [19] are generally attributed to xylem or phloem vessel plugging and/or systemic toxicity. The plugging theory indicates that the vascular vessels of infected plants are obstructed by fungal hyphae, thus limiting water transport in the xylem.

F. Molecular analyses
The bacterial colonies associated with the fungal hyphae in PDA plates were carefully isolated, single-colony purified and individual colonies identified using PCR. Molecular analyses (Fig. 4 d) indicated three specific bacteria viz., Pseudomonas fluorescens, Stenotrophomonas maltophila, and Paenibacillus polymyxa to be the ectosymbionts found on the hyphae of wilt causal Fusarium oxysporum wild types in healthy looking plants (Fig 4 F, G) but these ectosymbionts were absent in Fusarium hyphae of wilt disease affected field zone (Fig. 4 e). The ectosymbiont bacterium attached to the fungal hyphal surface may have also its own circadian clock mechanisms, in which bacterial cell signalling systems that detect blue photons within the visible light spectrum via a class of flavin-binding photosensors known as Light, Oxygen, Voltage (LOV) domains [20]. In covered soils, particularly just beneath the proximal top surface, such circadian factors can be expected to regulate the growth, reproduction cycles in a different manner to those of uncovered soils. In our study, the inoculant bacteria (Paenibacillus spp) agent appears to have acted as a biological control agent [21] and unsuspectingly via apparent selective attachment to the hyphae as ectosymbionts may have possibly promoted "virulence silencing mechanisms" in F. oxysporum [e.g. 22].
We have summarised an overview of our present study (Fig. 4 e-h) and its visual impacts. Light manipulation in horticulture crops using variable wavelength lighting is increasing interest within the horticulture business and industry [23]. The light regulation of metabolic pathways of fungi is now a major carrier of adaptation signalling information [24] for their interactions with host cells. To this end, light sensing is crucial to host plant and the fungi it interacts with as well as the bacteria that reside on the fungal hyphae, on account of the hyphal resident ectosymbiont bacterial secretion factors that are well documented to contribute to regulation of fungal pathogenicity and virulence mechanisms on host plants. Although not by any means complete, and requiring further studies employing in vivo molecular expression analyses, our soil covering operation manipulate light and temperature regimes and visibly (Fig. 4) influenced the outcome of pathogen-plant interactions. Plant stem / emerging roots interface establish holistic relationships with diverse microbial community in soil [25], nowadays known as "plant microbiome" i.e. total microbiota of plants [26] immensely impact the overall pathogen and disease control strategies. Given that, our results indicating that the soil covering promoted pathogen suppression via the healthy looking plants (Fig. 4 f, g) offers a practicable tool to the challenge of transposing the science of physiological molecular plant pathology to a tangible biotechnological solution on the ground from a farmer's perspective.

G. Impact of physiological factors (light, temperature) and microbial interventions
Our combinatorial data upon microbiological, SEM and preliminary molecular analyses suggest that Paenibacillus polymyxa followed by Pseudomonas spp., to be the bacteria most frequently found attached to the Fusarium hyphae that penetrated the M incana roots. In order to ascertain if these consortia of bacteria attached to fungal hyphae imposed any influence on the pathogenicity of Fusarium, the hyphae of bacteria were cured by repeated subculture on PDA supplemented with the antibiotic oxytetracycline (250µg /ml) and SEM confirmed that they were rendered virulent by virtue of penetrating plant root cell structures (Fig. 4 b, c). Our data obtained on bacterial ectosymbionts of Fusarium oxysporum hyphae from the rhizosphere of the host plant M. incana concurs with those of previous research [e.g. 27] on alternative horticultural crops such as lettuce. This is in agreement with current knowledge that diverse bacteria inhabit as fungal hyphal symbionts over a diverse range of phylogenetic variations [28]. Complex mechanisms of antibiosis, circadian clock phenomena, overarching cell signal proteins, Type III secretions, exudates, metabolite exchange, chemotaxis, microbial cross-talking (quorum sensing), symbiotic, host pathogenicity/virulence determination [29] including Fusarium oxysporum, these fungi-bacteria symbiosis are not exclusive to specific hosts. This microbial warfare happen as a rule in the overall development of host-pathogenic relationships in horticulture crops. Our microbiological data suggests that when wilt Fusarium hyphae are predisposed to inoculant biocontrol agent (e.g. Paenibacillus polumyxa) via the dip and drench treatments, the apparent virulence regulation effects expected may well lead to enhanced biological control of wilt disease. Further studies on light dependent gene expression analyses using latest real-time molecular tools may be required to monitor this environmental sensory perception of MHPRZ on both pathogenicity (wilt causal agent) and the virulence regulation (imposed by ectosymbiont on phytopathogenic fungal hyphae) in covered / uncovered regimes.

H. Effects of soil covering, thermal variations and influence on soil fungal populations
Results based on microbiological observations such as colony colour and hyphal features in the PDA plate and subsequent molecular confirmatory assays of a doctoral thesis in our laboratory [30] indicated that both covered and uncovered flower growers' soil bed samples harbour Fusarium oxysporum, F. venenatum F. culmorum and F. equiseti as the main Fusaria ( Table 2). The types of soil saprophytic fungi we identified corroborated other molecular investigations [31] to discriminate Fusarium spp. that are common soil inhabiting phytopathogens of small grain cereals, wheat and barley crops in Ireland soils [32]. The generic population density and their dynamics may also be attributed to the reduction of ambient temperature at both the surface and the top 2-5 cm soil depth following covering of the soil, compared to the uncovered treatments. The native Fusarium spp. populations exhibited ca. ~2.0 x 10 4 colony forming units (CFUs) g -1 of either covered or uncovered soils that were widely prevalent in the Northern Ireland polytunnel floriculture soil-bed. The wilt causal fungi Fusarium oxysporum (wild types) occurred at a higher population density of 2.4 x 10 5 CFUs g -1 in uncovered soils, and there was a marked reduction of two log10 folds to 3.4 x 10 3 CFUs g -1 when the soils were covered by polythene. Nonpathogenic F. oxysporum isolates commonly occur in the soil as saprophytes, while some have been identified as biocontrol agents and endophytes [33].

I. Soil as a natural reservoir for non-pathogenic fungal antagonists against wilt Fusarium
In this present study, it was intriguing to note the widespread prevalence of a natural reservoir of other fungal antagonists ( Table 2) such as Trichoderma atroviride formerly T. harzianum [34], T. citrinoviride, Eurotiomyces spp., Clonostachys pseudochrolueca [35] which possess inhibitory potential towards Fusarium populations. Some of the above fungal-fungal antagonists are registered as commercial formulations in Europe [36] as wilt fungal disease suppressants. In vitro inhibitory assay (Table 3) revealed that the Fusarium spp achieved a moderate (~20%) suppression of local wilt causal isolates of F. oxysporum, while other fellow native soil fungi (Gliocadium, Chlonostachys and Trichoderma species) in the soils were significantly stronger ( > 60%) inhibitors of the local flower wilt Fusaria. Wilt antagonistic fungi [Trichoderma atroviride, T. citrinoviride, Eurotiomyces spp., Clonostachys pseudochrolueca] increased by at least 1.0 x log10 fold from ca <1.0 x 10 2 CFUs g -1 in uncovered soils to ~3.2 x 10 3 CFUs g -1 when covered with polythene sheets. Such small but significant (p=0.01) shifts in fungal population densities were noticeable after physiological manipulation of covering the soil than their counterpart uncovered soil plots which exhibited lower abundance of co-colonising wilt antagonistic fungal populations. The soil covering operation in our study is quite encouraging in respect of the same serving as a natural means of supplementing the biological agents commonly used for suppression of wilt disease and offers low cost biosecurity options for the cut-flower growing rural entrepreneurs in challenging competitive farming markets for Ireland within Europe. Our findings that varying environmental conditions and thereby enhancing the prospects of non-pathogenic natural biological control fungal agents inhabitants in soil themselves for suppression of Fusarium wilt diseases concurs in some respects with previous attempts to manipulate cropping conditions, soil type and Fusarium oxysporum inhibitory fungi [37], and as alternative disease management strategy in greenhouses for control of Fusarium wilt of tomato [38].  [42], and thus most likely to be of native in origin, confined to the local Northern Ireland soils within the island of Ireland. Our molecular analyses (Fig 4 D) concurred with the conclusions of another study in Northern Italy in that, the Italian researchers seminal work [43] and later using molecular characterization [44] through IGS sequencing demonstrated that the forma specialis matthioli (ATCC16602 and ATCC16603) were not pathogenic on lamb's lettuce and isolates of different origins differed genetically from isolates that were poorly, moderately or highly virulent.
Due to the fact that both lettuce and cut flower picking involves phytosanitory considerations, transfer of infested soil through trays, pallets and the foot wear of farm workers has been confirmed as the main route that the disease lettuce Fusarium wilt as well those encountered in cut flower wilts are spread. In another study in Florida, researchers [45] found that due to the limitations of land availability for fallowing and rotation, cut flower growers have to contend with a number of other soil-borne wilt causal agents viz., Pythium and Rhizoctonia apart from Fusarium menace. These phytopathogenic species are present throughout their cultivation cycles in the year and are unresponsive to routine soil sterilisation. However, given that in our study, the grower had imposed stringent soil disinfection measures, as well as covering the soil with polythene sheets, the impact of the intercropping and human foot spread of Fusarium wilt may be subdued. Alternative biologically sustainable methods are increasingly becoming popular with farmers such as corn crop liquor [46] for lettuce root rot, new range of biostimulants also known as 'bioeffectors' [47], [48] comprising plant growth promoting, pathogen controlling bacteria, fungi and processed macroalgae extracts for crop management to reduce increasing climate change driven abiotic & biotic stresses [47], [48].

IV. CONCLUSION
Fundamental molecular mechanisms of saprophytic fungal spore adaptation, growth physiology and host pathogenicity are innate fungal behaviours. To translate this science and transfer the knowledge into 'practicable biotechnological solutions' for ornamental (cut flower) crops disease management by farmers is a challenge for agricultural scientists. To this end, our present work demonstrated simple agricultural practices such as physiological manipulation of temperature and light via covering of soil and interactive use of an enrichment compendium of native microbe as biocontrol agents themselves isolated from the disease prone soils for poly tunnel farming offer plausible solution to horticulturists. Our results also indicate the sustainable potential interventions to increase the resilience of horticulture and floriculture crops against recurrent persistent soil inhabiting pathogen opportunism and infection control.