Adapting the inoculation methods of kiwifruit canker disease to identify efficient biocontrol bacteria from branch microbiome.

Pseudomonas syringae pv. actinidiae (Psa), the bacterium that causes kiwifruit bacterial canker, is a common field occurrence that is difficult to control globally. Currently, exploring the resources for efficient biocontrol bacteria is a hot spot in the field. The common strategy for isolating biocontrol bacteria is to directly isolate biocontrol bacteria that can secrete diffusible antibacterial substances, most of which are members of Bacillus, Pseudomonas and Streptomycetaceae, from disease samples or soil. Here, we report a new approach by adapting the typical isolation methods of kiwifruit canker disease to identify efficient biocontrol bacteria from the branch microbiome. Using this unique approach, we isolated a group of kiwifruit biocontrol agents (KBAs) from the branch microbiome of Psa-resistant varieties. Thirteen of these showed no antagonistic activity in vitro, which depends on the secretion of antibacterial compounds. However, they exhibited antibacterial activity via cell-to-cell contacts mimicked by co-culture on agar plates. Through biocontrol tests on plants, two isolates, KBA13 and KBA19, demonstrated their effectiveness by protecting kiwifruit branches from Psa infection. Using KBA19, identified as Pantoea endophytica, as a representative, we found that this bacterium uses the type VI secretion system (T6SS) as the main contact-dependent antibacterial weapon that acts via translocating toxic effector proteins into Psa cells to induce cell death, and that this capacity expressed by KBA19 is common to various Psa strains from different countries. Our findings highlight a new strategy to identify efficient biocontrol agents that use the T6SS to function in an antibacterial metabolite-independent manner to control wood diseases.

Assembly of an active microbial consortium by engineering compatible combinations containing foreign and native biocontrol bacteria of kiwifruit.

Assembling functional bacterial biocontrol consortia is expected to expand the scope and efficiency of biocontrol agents. Generally, bacterial interspecies interactions lead to incompatibility events, as bacteria can produce antibacterial compounds and/or assemble contact-dependent killing (CDK) devices. Here, we aimed to assemble a bacterial consortium comprising Lysobacter enzymogenes OH11 and Bacillus safensis ZK-1 for the synergistic control of bacterial and fungal diseases of kiwifruit. ZK-1, a native kiwifruit biocontrol bacterium, is effective against Pseudomonas syringae pv. actinidiae (Psa) that causes bacterial kiwifruit canker, but has weak antifungal activity. OH11 is a foreign kiwifruit biocontrol agent with strong antifungal activity. While OH11 was unable to produce anti-Gram-negative metabolites, this strain could utilize type IV secretion system as an antibacterial CDK weapon. We first observed that OH11 could inhibit growth of ZK-1 by generating diffusible anti-Gram-positive antibiotic WAP-8294A2, whereas ZK-1 failed to generate diffusible antibacterial compound to inhibit growth of OH11. To disrupt this interspecies incompatibility, we generated a transgenic OH11-derived strain, OH11W, by deleting the WAP-8294A2 biosynthetic gene and found that OH11W did not kill ZK-1. We further observed that when OH11W and ZK-1 were co-inoculated on agar plates, no CDK effect was observed between them, whereas co-culture of OH11W or ZK-1 with Psa on agar plates resulted in Psa killing, suggesting L. enzymogenes and B. safensis assemble antibacterial CDK weapons against bacterial pathogens, and these CDK weapons did not affect the compatibility between OH11W and ZK-1. Based on these findings, we assembled an OH11W/ZK-1 dependent consortium that was shown to be functional in controlling bacterial canker and several representative fungal diseases of kiwifruit.

Effects of Short-Term High Temperature on Gas Exchange in Kiwifruits (Actinidia spp.).

Climate changes occurring today require detailed research of the effects of increasing temperatures on photosynthesis in different species and cultivars. Temperature variability is a crucial determinant of the yield and quality of plants, particularly when high-temperature episodes coincide with their growth and fruit development. The effect of high temperature (HT) on higher plants primarily concerns photosynthetic functions, but the sensitivity of photosynthesis to high temperature is not well-understood in kiwifruits. In this study, we designed a new environmental monitoring system to evaluate the effects of environmental factors on the photosynthetic physiology of different kiwifruit species and cultivars. A significant positive correlation was determined between the main photosynthetic indices of kiwifruits, such as transpiration rate and net photosynthetic rate. The net photosynthetic rate of commercial kiwifruit cultivars was strongly inhibited when the temperature exceeded 44.5 °C, and the leaves of kiwifruits were irreversibly damaged when the temperature increased to 52 °C. Kiwifruit cultivars with different ploidy levels (diploid, tetraploid and hexaploid) were found to be sensitive to high temperature, whereas tetraploids had higher HT resistance and hexaploids had the highest net photosynthetic rate. Further research showed that the HT tolerance of kiwifruits existed not only between species but also among cultivars. A. eriantha had the highest net photosynthetic rate at more than 44.7 °C, but those of A. deliciosa and A. arguta declined sharply as the temperature exceeded 43.5 °C. As a result, it was determined that high temperatures have important effects on the photosynthetic activities of kiwifruit plants with different ploidy levels, and that these effects can significantly change their development according to how they differ among different species/cultivars.

Characterizing Tetraploid Populations of Actinidia chinensis for Kiwifruit Genetic Improvement.

Understanding genetic diversity and structure in natural populations and their suitable habitat response to environmental changes is critical for the protection and utilization of germplasm resources. We evaluated the genetic diversity and structure of 24 A. chinensis populations using simple sequence repeat (SSR) molecular markers. The potential suitable distribution of tetraploid A. chinensis estimated under the current climate and predicted for the future climate was generated with ecological niche modeling (ENM). The results indicated that the polyploid populations of A.chinensis have high levels of genetic diversity and that there are distinct eastern and western genetic clusters. The population structure of A. chinensis can be explained by an isolation-by-distance model. The results also revealed that potentially suitable areas of tetraploids will likely be gradually lost and the habitat will likely be increasingly fragmented in the future. This study provides an extensive overview of tetraploid A. chinensis across its distribution range, contributing to a better understanding of its germplasm resources. These results can also provide the scientific basis for the protection and sustainable utilization of kiwifruit wild resources.

Kiwifruit MYBS1-like and GBF3 transcription factors influence l-ascorbic acid biosynthesis by activating transcription of GDP-L-galactose phosphorylase 3.

Plant-derived Vitamin C (l-ascorbic acid (AsA)) is crucial for human health and wellbeing and thus increasing AsA content is of interest to plant breeders. In plants GDP-l-galactose phosphorylase (GGP) is a key biosynthetic control step and here evidence is presented for two new transcriptional activators of GGP. AsA measurement, transcriptomics, transient expression, hormone application, gene editing, yeast 1/2-hybrid, and electromobility shift assay (EMSA) methods were used to identify two positively regulating transcription factors. AceGGP3 was identified as the most highly expressed GGP in Actinidia eriantha fruit, which has high fruit AsA. A gene encoding a 1R-subtype myeloblastosis (MYB) protein, AceMYBS1, was found to bind the AceGGP3 promoter and activate its expression. Overexpression and gene-editing show AceMYBS1 effectively increases AsA accumulation. The bZIP transcription factor AceGBF3 (a G-box binding factor), also was shown to increase AsA content, and was confirmed to interact with AceMYBS1. Co-expression experiments showed that AceMYBS1 and AceGBF3 additively promoted AceGGP3 expression. Furthermore, AceMYBS1, but not GBF3, was repressed by abscisic acid, resulting in reduced AceGGP3 expression and accumulation of AsA. This study sheds new light on the roles of MYBS1 homologues and ABA in modulating AsA synthesis, and adds to the understanding of mechanisms underlying AsA accumulation.

Cytotype distribution and chloroplast phylogeography of the Actinidia chinensis complex.

BACKGROUND: Plant phylogeographic studies of species in subtropical China have mainly focused on rare and endangered species, whereas few studies have been conducted on taxa with relatively wide distribution, especially polyploid species. We investigated the cytotype and haplotype distribution pattern of the Actinidia chinensis complex, a widespread geographically woody liana with variable ploidy in subtropical China comprising two varieties, with three chloroplast fragments DNA (ndhF-rpl132, rps16-trnQ and trnE-trnT). Macroevolutionary, microevolutionary and niche modeling tools were also combined to disentangle the origin and the demographic history of the species or cytotypes. RESULTS: The ploidy levels of 3338 individuals from 128 populations sampled throughout the species distribution range were estimated with flow cytometry. The widespread cytotypes were diploids followed by tetraploids and hexaploids, whereas triploids and octoploids occurred in a few populations. Thirty-one chloroplast haplotypes were detected. The genetic diversity and genetic structure were found to be high between varieties (or ploidy races) chinensis and deliciosa. Our results revealed that these two varieties inhabit significantly different climatic niche spaces. Ecological niche models (ENMs) indicate that all varieties' ranges contracted during the Last Inter Glacial (LIG), and expanded eastward or northward during the Last Glacial Maximum (LGM). CONCLUSIONS: Pliocene and Plio-Pleistocene climatic fluctuations and vicariance appear to have played key roles in shaping current population structure and historical demography in the A. chinensis complex. The polyploidization process also appears to have played an important role in the historical demography of the complex through improving their adaptability to environmental changes.

ABE8e with Polycistronic tRNA-gRNA Expression Cassette Sig-Nificantly Improves Adenine Base Editing Efficiency in Nicotiana benthamiana.

Adenine base editor containing TadA8e (ABE8e) has been reported in rice. However, the application of ABE8e in other plant species has not been described, and the comparison between ABE8e and ABE7.10, which is widely used in plants, has also been poorly studied. Here, we developed the ABE8e with the polycistronic tRNA-gRNA expression cassette (PTG-ABE8e) and PTG-ABE7.10 and compared their A-to-G editing efficiencies using both transient and stable transformation in the allotetraploid Nicotiana benthamiana. We found that the editing efficiency of PTG-ABE8e was significantly higher than that of PTG-ABE7.10, indicating that ABE8e was more efficient for A-to-G conversion in N. benthamiana. We further optimized the ABE8e editing efficiency by changing the sgRNA expression cassette and demonstrated that both PTG and single transcript unit (STU) enhanced ABE8e efficiency for A-to-G conversion in N. benthamiana. We also estimated the potential off-target effect of PTG-ABE8e at potential off-targeting sites predicted using an online tool in transgenic plants, and no off-target editing event was found for potential off-targeting sites selected, indicating that ABE8e could specifically facilitate A-to-G conversion. Our results showed that ABE8e with PTG structure was more suitable for A-to-G conversion in N. benthamiana and provided valuable clues for optimizing ABE tools in other plants.

First Report of Bacterial Leaf Spot Disease of Broussonetia papyrifera Caused by Pseudomonas syringae pv. actinidiae in China.

Broussonetia papyrifera (paper mulberry) is a deciduous tree with a number of uses and is native to northeastern Asia. Because of its fast-growing nature and high tolerance to dust, smoke, and high temperatures, paper mulberry is regarded as an important and economically-valuable component of a biologically diverse community and is used extensively in several areas including medicine, animal husbandry, paper making, weaving, afforestation and light industry (Mei et al. 2016). From June to August of 2019, symptoms on paper mulberry trees were observed in Shiniushan village, Sanhua town, Xishui County, Hubei province of China. Typical symptoms on leaves included small, angular, brown spots surrounded by yellow haloes. These spots coalesced into necrotic areas. The incidence was around 30%, which threatened the survival and reduced the yield of paper mulberry. In order to identify the causal pathogenic organism, leaf samples from 10 different infected trees were collected every two weeks and isolations made over three months. Several circular, flat, granulated colonies with entire margins were isolated on King's B medium (KB). The biochemical and physiological characteristics of thirty typical strains were tested and listed as following: gram negative, aerobic, rod shaped, and non-fluorescent on King's B medium; positive for carbohydrate utilization (sucrose, glucose, fructose and arabinose), levan production, hypersensitive on tobacco, potato and tomato; and negative for oxidase, arginine dehydrolase, tyrosinase and urease activity, gelatin liquefaction, and reduction of nitrate. Psa pathovar-specific primers PsaF1/PsaR2 (280bp product ) identified two representative strains as Psa (Rees-George et al. 2010). BLAST analysis further confirmed that the 16S rDNA region amplified by primers 27F/1492R (NCBI accession nos. MT472100 and MT472101) shared 99.84% and 99.77% identity with the Psa type strain ICMP 18884 (CP011972) respectively (Weisburg et al. 1991). For ten typical strains, pathogenicity was confirmed by spraying a bacterial suspension (108 cfu/mL) onto fifty one-year seedlings of B. papyrifera, five seedlings repetitions for each strain. Symptoms of infection similar to those observed initially in the field were detected within 7 days after incubation at 25°C with 80-85% humidity. No symptoms were observed on control plants. The pathogen was re-isolated from symptomatic leaves and re-identified as Psa by morphological characteristics and sequencing. To our knowledge, this is the first report of Psa causing bacterial leaf spot disease on B. papyrifera, China. Psa has been reported as a pathogen causing bacterial canker of kiwifruit worldwide, resulting in severe economic losses to kiwifruit growers (McCann & Li, 2017). As a host of Psa, B. papyrifera may be a source of inoculum for nearby kiwifruit orchards, and consequently effective control measures should be taken to manage this disease. Funding: This study was supported by the National Natural Science Foundation of China (31701974; 31901980), Science and technology program funded by Wuhan Science and Technology Bureau (2018020401011307). References: Mei et al. 2016. Eur J Plant Pathol. 145: 203. McCann & Li et al. 2017. Genome Biol Evol. 9: 932. Rees-George et al. 2010. Plant Pathol. 59: 453 Weisburg et al. 1991. J Bacteriol. 173: 697.

Volatile fingerprints and biomarkers of three representative kiwifruit cultivars obtained by headspace solid-phase microextraction gas chromatography mass spectrometry and chemometrics.

Volatile aroma of kiwifruit is a mixture of complicated and time-dependent compounds, and thereby the study of these compounds required distinguished analytical techniques as well as robust data analysis techniques. In this work, we report on the volatile fingerprints and biomarkers of three representative kiwifruit cultivars with commercial importance using headspace solid-phase microextraction gas chromatography-mass spectrometric (HS-SPME-GC-MS) coupled with multivariate analysis. As a result, 95 volatiles have been analyzed from the fingerprints, and ultimately six of which were identified as volatile biomarkers of the kiwifruit cultivars studied, which are formic acid octyl ester, 2-Methylbicyclo[4.3.0]non-1(6)-ene, 1-ethoxy-2,4-hexadiene, and 2-methyl-5-(1-methylethyl)-bicyclo[3.1.0]hex-2-ene for Jintao (A. chinensis), and 1-methoxy-2-methyl-benzene and (E,E)-2,4-heptadienal for Cuiyu (A. deliciosa), respectively. Since the samples of each cultivar were in various maturities, these compounds could be taken as the maturity-independent volatile biomarkers for the kiwifruit cultivars, which would be valuable for marker-assisted flavour breeding in the kiwifruit production.

Whole transcriptome sequencing of Pseudomonas syringae pv. actinidiae-infected kiwifruit plants reveals species-specific interaction between long non-coding RNA and coding genes.

An outbreak of kiwifruit bacterial canker disease caused by Pseudomonas syringae pv. actinidiae (Psa) beginning in 2008 caused disaster to the kiwifruit industry. However the mechanisms of interaction between kiwifruit and Psa are unknown. Long noncoding RNAs (lncRNAs) are known to regulate many biological processes, but comprehensive repertoires of kiwifruit lncRNAs and their effects on the interaction between kiwifruit and Psa are unknown. Here, based on in-depth transcriptomic analysis of four kiwifruit materials at three stages of infection with Psa, we identified 14,845 transcripts from 12,280 loci as putative lncRNAs. Hierarchical clustering analysis of differentially-expressed transcripts reveals that both protein-coding and lncRNA transcripts are expressed species-specifically. Comparing differentially-expressed transcripts from different species, variations in pattern-triggered immunity (PTI) were the main causes of species-specific responses to infection by Psa. Using weighted gene co-expression network analysis, we identified species-specific expressed key lncRNAs which were closely related to plant immune response and signal transduction. Our results illustrate that different kiwifruit species employ multiple different plant immunity layers to fight against Psa infection, which causes distinct responses. We also discovered that lncRNAs might affect kiwifruit responses to Psa infection, indicating that both protein-coding regions and noncoding regions can affect kiwifruit response to Psa infection.

Agronomic Trait Variations and Ploidy Differentiation of Kiwiberries in Northwest China: Implication for Breeding.

Polyploid plants often have higher biomass and superior crop qualities. Breeders therefore search for crop germplasm with higher ploidy levels; however, whether higher ploidy levels are associated with better performance remains unclear. Actinidia arguta and related species, whose commercialized fruit are referred to as kiwiberries, harbor a series of ploidy races in nature, offering an opportunity to determine the link between ploidy levels and agronomic traits. In the present study, we determined the ploidy levels of A. arguta var. arguta, A. arguta var. giraldii, and A. melanandra in 16 natural populations using flow cytometry, and examined 31 trait variations in fruits, leaves and flowers by field observations, microscopic examination and laboratory analyses. Our results showed that octaploid and decaploid A. arguta var. giraldii had larger dimension of leaves than tetraploid A. arguta var. arguta and A. melanandra, but their fruits were significantly smaller. In addition, A. arguta var. giraldii (8x and 10x) had higher contents of nutrients such as ascorbic acid and amino acids; however, some important agronomic traits, including the content of total sugar and total acid, were significantly lower in the octaploids and decaploids. Moreover, octaploids and decaploids did not result in greater ecological adaptability for the challenging environments and climates. In conclusion, the differentiation of ecological adaptability and traits among natural kiwiberries' cytotypes suggested that higher ploidy levels are not inevitably advantageous in plants. The findings of A. arguta and related taxa in geographical distribution and agronomic trait variations will facilitate their germplasm domestication.

Origin and Evolution of the Kiwifruit Canker Pandemic.

Recurring epidemics of kiwifruit (Actinidia spp.) bleeding canker disease are caused by Pseudomonas syringae pv. actinidiae (Psa). In order to strengthen understanding of population structure, phylogeography, and evolutionary dynamics, we isolated Pseudomonas from cultivated and wild kiwifruit across six provinces in China. Based on the analysis of 80 sequenced Psa genomes, we show that China is the origin of the pandemic lineage but that strain diversity in China is confined to just a single clade. In contrast, Korea and Japan harbor strains from multiple clades. Distinct independent transmission events marked introduction of the pandemic lineage into New Zealand, Chile, Europe, Korea, and Japan. Despite high similarity within the core genome and minimal impact of within-clade recombination, we observed extensive variation even within the single clade from which the global pandemic arose.

Analysis of volatile compounds responsible for kiwifruit aroma by desiccated headspace gas chromatography-mass spectrometry.

A new method for desiccated headspace (DHS) sampling of aqueous sample to GC-MS for the analysis of volatile compounds responsible for kiwifruit aroma in different kiwifruit cultivars has been developed based on the complete hydrate formation between the sample solvent (water) with anhydrous salt (calcium chloride) at an elevated temperature (above the boiling point of the aqueous sample) in a non-contact format, which overcame the water-effect challenge to directly introduce aqueous sample into GC-MS analysis. By means of DHS, the volatile compounds in three different kiwifruit cultivars were analyzed and compared under the optimized operating conditions, mainly time and temperature for headspace equilibration, column temperature program for GC-MS measurement. As a result, 20 peaks of volatile compounds responsible for kiwifruit aroma were detected and remarkable differences were found in the relative contents of three major volatile compounds among the three different kiwifruit cultivars, i.e., acetaldehyde, ethanol and furfural. The DHS sampling technique used in the present method can make the GC-MS analysis of volatile compounds in the aqueous sample within complex matrix possible without contaminating the GC-MS instrument. In terms of the analysis of volatile compounds in kiwifruit, the present method enabled a direct measurement on the filtrate of the aqueous kiwifruit pulp, without intermediate trap phase for the extraction of analytes, which will be more reliable and simpler as compared with any other headspace method in use. Thus, DHS coupled with GC-MS will be a new valuable tool available for the kiwifruit related research and organoleptic quality control.

The molecular diversity analysis of Auricularia auricula-judae in China by nuclear ribosomal DNA intergenic spacer

Background: For the crossbreeding of Auricularia auricula-judae, selecting the appropriated parents in hybridization is very important. However, the classification and diversity analysis of A. auricula-judae has been equivocal, due to the similarity of the fruiting body morphology and its susceptibility to environmental influences. For this purpose, the molecular diversity of 32 A. auricula-judae commercial cultivars in China was analyzed by using the nuclear ribosomal DNA intergenic spacer. Results: The complete nuclear rDNA gene complex of A. auricula-judae isolate is 11,210 bp long, and contains the 18S, 5.8S, and 28S rRNA gene as well as the ITS and IGS regions. Based on the sequence data, four more effective primer combinations for the IGS region of A. auricula-judae were designed. Nucleotide sequence variation in the IGS among 32 A. auricula-judae commercial cultivars in China sorted into three strongly supported clades, which is correlated with geographical regions. Most strains originated from the same area were with a narrow genetic basis and could possibly be domesticated from the local wild-type strains. Conclusion: The grouping information obtained in the present work provides significant information for further genetic improvement in A. auricula-judae, and suggested that the IGS region can be used as an excellent tool for identification of genetic variation.