in-vivo-leaf-inoculation-an-alternative-method-to-assess-disease

In Vivo Leaf Inoculation: An Alternative Method to Assess the Disease Resistance of Hybrid Clones in Poplar Breeding of Stem Canker Disease

Stem canker diseases caused by the pathogen Cytospora chrysosperma (Pers.) Fr.) and Botryosphaeria dothidea (Moug. ex Fr.) Ces. &#38; de Not. are the two ...

chinese academy of forestry

Comprehensive Experimental Center in Yellow River Delta of Chinese Academy of Forestry

Spathius agrili Yang (Hymenoptera: Braconidae) is a gregarious larval ectoparasitoid of the emerald ash borer, Agrilus planipennis Fairmaire (Coleoptera: Buprestidae) and is a recently described species. Both pest and parasitoid are native to China. In Tianjin City, China, S. agrili typically exhibited 3-4 generations per year, overwintering as a prepupa in a cocoon inside the host gallery. The multiple generations of S. agrili overlapped with its host, as did the emergence dates of the overwintering generation. From a single host, 1-18 S. agrili successfully developed to the adult stage (average 8.4), but in all cases the host was killed. The sex ratio (female: male) of the parasitoid adults emerging from field-collected cocoons was 2:1, whereas the sex ratio of parasitoids reared from field collected eggs and larvae was greater than 3:1. On average, adult females lived 29.1 d, and males lived 23.6 d when fed with 20% honey solution, significantly longer than without a nutritional supplement. Sexual reproduction is the normal mode of reproduction, but in the laboratory females did reproduce parthenogenetically, producing only males. The average fecundity was 23.3 eggs per female in the laboratory. S. agrili developed through five larval instars, and the larvae fed gregariously on the host hemolymph. The generation time from egg to adult wasp was 27-28 d at 22-26 degrees C. Natural parasitism rates were as high as 60%, and in October they reached over 90% in some stands. This study showed that S. agrili is a promising agent for biocontrol of A. planipennis.

Biology and behavior of Spathius agrili, a parasitoid of the emerald ash borer, Agrilus planipennis, in China.

The biology, ecology, and life cycle of the emerald ash borer, Agrilus planipennis Fairmaire (Coleoptera: Buprestidae), were studied using regular inspection in the forest and observations in the laboratory. Results indicated that A. planipennis are mostly univoltine in Tianjin, China. They overwintered individually as mature larvae in shallow chambers excavated in the outer sapwood. In late July, some full-grown larvae began to build overwintering chambers, and all larvae entered the sapwood for dormancy by early November. A. planipennis pupated in the overwintering chamber from early April to mid May the following year, and the average pupal duration was about 20 days. In late April, some newly eclosed adults could be found in the pupal cells, but they had not yet emerged from the tree. Adults began to emerge in early May, with peak flight occurring in mid May. The average longevity of adults was about 21 days and the adult stage lasted through early July. The adults fed on ash foliage as a source of nutrition. Mating was usually conducted and completed on the leaf or trunk surfaces of ash trees. Oviposition began in mid May and eggs hatched on average in 15.7 days. The first instar larvae appeared in early June. The larval stage lasted about 300 days to complete an entire generation. The emerald ash borer had four larval instars on velvet ash, Fraxinus velutina (Scrophulariales: Oleaceae). The major natural control factors of A. planipennis were also investigated, and preliminary suggestions for its integrated management are proposed.

The biology and ecology of the emerald ash borer, Agrilus planipennis, in China.

We investigated the effects of salt-sensitive signaling molecules on ionic fluxes and gene expression related to K/Na homeostasis in a perennial herb, , during short-term NaCl stress (100 mM, 24 h). Salt treatment caused more pronounced Na accumulation in root cells than in leaf cells. Na ions were mostly compartmentalized in vacuoles. Roots exposed to NaCl showed increased levels of extracellular ATP (eATP), cytosolic Ca, HO, and NO. Steady-state flux recordings revealed that these salt-sensitive signaling molecules enhanced NaCl-responsive Na efflux, due to the activated Na/H antiport system in the plasma membrane (PM). Moreover, salt-elicited K efflux, which was mediated by depolarization-activated cation channels, was reduced with the addition of Ca, HO, NO, and eATP. The salt-adaptive effects of these molecules (Na extrusion and K maintenance) were reduced by pharmacological agents, including LaCl (a PM Ca channel inhibitor), DMTU (a reactive oxygen species scavenger), cPTIO (an NO scavenger), or PPADS (an antagonist of animal PM purine P2 receptors). RT-qPCR data showed that the activation of the PM Na/H antiport system in salinized roots most likely resulted from the upregulation of two genes,  and , which encoded the PM Na/H antiporter, salt overly sensitive 1 (SOS1), and H-ATPase, respectively. Clear interactions occurred between these salt-sensitive agonists to accelerate transcription of salt-responsive signaling pathway genes in  roots. For example, Ca, HO, NO, and eATP promoted transcription of  (salt overly sensitive 3) and/or  (CBL-interacting protein kinase) to activate the predominant Ca-SOS signaling pathway in salinized liquorice roots. eATP, a novel player in the salt response of , increased the transcription of ,  (respiratory burst oxidase homolog protein D),  (nitrate reductase), , and  (the mitogen-activated protein kinases 3 and 6). Moreover,  and  expression levels were enhanced by HO in NaCl-stressed  roots. Our results indicated that eATP triggered downstream components and interacted with Ca, HO, and NO signaling to maintain K/Na homeostasis. We propose that a multiple signaling network regulated K/Na homeostasis in NaCl-stressed  roots.

Salt-Sensitive Signaling Networks in the Mediation of K/Na Homeostasis Gene Expression in  Roots.

CDPKs (Ca-Dependent Protein Kinases) are very important regulators in plant response to abiotic stress. The molecular regulatory mechanism of CDPKs involved in salt stress tolerance remains unclear, although some CDPKs have been identified in salt-stress signaling. Here, we investigated the function of an  CDPK, CPK12, in salt-stress signaling. The -RNA interference (RNAi) mutant was much more sensitive to salt stress than the wild-type plant GL1 in terms of seedling growth. Under NaCl treatment, Na⁺ levels in the roots of -RNAi plants increased and were higher than levels in GL1 plants. In addition, the level of salt-elicited H₂O₂ production was higher in -RNAi mutants than in wild-type GL1 plants after NaCl treatment. Collectively, our results suggest that CPK12 is required for plant adaptation to salt stress.

The Arabidopsis Ca-Dependent Protein Kinase CPK12 Is Involved in Plant Response to Salt Stress.

Sodium chloride (NaCl) induced expression of a jacalin-related mannose-binding lectin () gene in leaves, roots, and callus cultures of  (salt-resistant poplar). To explore the mechanism of the  in salinity tolerance, the full length of  was cloned from  and was transformed into Arabidopsis. PeJRL was localized to the cytoplasm in mesophyll cells. Overexpression of  in Arabidopsis significantly improved the salt tolerance of transgenic plants, in terms of seed germination, root growth, and electrolyte leakage during seedling establishment. Under NaCl stress, transgenic plants retained K⁺ and limited the accumulation of Na⁺. -transgenic lines increased Na⁺ extrusion, which was associated with the upward regulation of , , and  genes encoding plasma membrane Na⁺/proton (H⁺) antiporter and H⁺-pumps. The activated H⁺-ATPases in -overexpressed plants restricted the channel-mediated loss of K⁺ that was activated by NaCl-induced depolarization. Under salt stress, ⁻transgenic Arabidopsis maintained reactive oxygen species (ROS) homeostasis by activating the antioxidant enzymes and reducing the production of O₂ through downregulation of NADPH oxidases. Of note, the transgenic Arabidopsis repressed abscisic acid (ABA) biosynthesis, thus reducing the ABA-elicited ROS production and the oxidative damage during the period of salt stress. A schematic model was proposed to show the mediation of PeJRL on ABA response, and ionic and ROS homeostasis under NaCl stress.

 JRL Mediates ABA Response, Ionic and ROS Homeostasis in Arabidopsis under Salt Stress.

Carbon starvation is the current leading hypothesis of plant mortality mechanisms under drought stress; recently, it is also used to explain tree die-off in plant diseases. However, the molecular biology of the carbon starvation pathway is unclear. Here, using a punch inoculation system, we conducted transcriptome and physiological assays to investigate pathogen response in poplar stems at the early stages of Botryosphaeria and Valsa canker diseases. Transcriptome assays showed that the majority of differentially expressed genes (DEGs) in stem phloem and xylem, such as genes involved in carbon metabolism and transportation, aquaporin genes (in xylem) and genes related to the biosynthesis of secondary metabolites and the phenylpropanoid pathway (related to lignin synthesis), were downregulated at 7 days after inoculation (DAI). Results also showed that the expression of the majority of disease-resistance genes upregulated in poplar stems, which may be connected with the downregulation expression of the majority of WRKY family genes. Physiological assays showed that transpiration rate decreased but WUE (water use efficiency) increased the 3 and 7 DAI, while the net photosynthetic rate decreased at 11 DAI in Botryosphaeria infected poplars (ANOVA, P < 0.05). The NSC (non-structural carbohydrates) content assays showed that the soluble sugar content of stem phloem samples increased at 3, 7, and 11 DAI that might due to the impede of pathogen infection. However, soluble sugar content of stem xylem and root samples decreased at 11 DAI; in contrast, the starch content unchanged. Therefore, results revealed a chronological order of carbon related molecular and physiological performance: declination of genes involved in carbon and starch metabolism first (at least at 7 DAI), declination of assimilation and carbon reserve (at 11 DAI) second. Results implied a potential mechanism that affects the host carbon reserve, by directly inhibiting the expression of genes involved in carbon metabolism and transport.

Fungal canker pathogens trigger carbon starvation by inhibiting carbon metabolism in poplar stems.

Plasma membrane proton pumps play a crucial role in maintaining ionic homeostasis in salt-resistant Populus euphratica under saline conditions. High levels of NaCl (200 mM) induced PeHA1 expression in P. euphratica roots and leaves. We isolated a 2022 bp promoter fragment upstream of the translational start of PeHA1 from P. euphratica. The promoter-reporter construct PeHA1-pro::GUS was transferred to tobacco plants, demonstrating that β-glucuronidase activities increased in root, leaf, and stem tissues under salt stress. DNA affinity purification sequencing revealed that PeWRKY1 protein targeted the PeHA1 gene. We assessed the salt-induced transcriptional response of PeWRKY1 and its interaction with PeHA1 in P. euphratica. PeWRKY1 binding to the PeHA1 W-box in the promoter region was verified by a yeast one-hybrid assay, EMSA, luciferase reporter assay, and virus-induced gene silencing. Transgenic tobacco plants overexpressing PeWRKY1 had improved expression of NtHA4, which has a cis-acting W-box in the regulatory region, and improved H+ pumping activity in both in vivo and in vitro assays. We conclude that salt stress up-regulated PeHA1 transcription due to the binding of PeWRKY1 to the W-box in the promoter region of PeHA1. Thus, we conclude that enhanced H+ pumping activity enabled salt-stressed plants to retain Na+ homeostasis.

Populus euphratica WRKY1 binds the promoter of H+-ATPase gene to enhance gene expression and salt tolerance.

Remorins (REMs) play an important role in the ability of plants to adapt to adverse environments. PeREM6.5, a protein of the REM family in Populus euphratica (salt-resistant poplar), was induced by NaCl stress in callus, roots and leaves. We cloned the full-length PeREM6.5 from P. euphratica and transformed it into Escherichia coli and Arabidopsis thaliana. PeREM6.5 recombinant protein significantly increased the H+-ATPase hydrolytic activity and H+ transport activity in P. euphratica plasma membrane (PM) vesicles. Yeast two-hybrid assay showed that P. euphratica REM6.5 interacted with RPM1-interacting protein 4 (PeRIN4). Notably, the PeREM6.5-induced increase in PM H+-ATPase activity was enhanced by PeRIN4 recombinant protein. Overexpression of PeREM6.5 in Arabidopsis significantly improved salt tolerance in transgenic plants in terms of survival rate, root growth, electrolyte leakage and malondialdehyde content. Arabidopsis plants overexpressing PeREM6.5 retained high PM H+-ATPase activity in both in vivo and in vitro assays. PeREM6.5-transgenic plants had reduced accumulation of Na+ due to the Na+ extrusion promoted by the H+-ATPases. Moreover, the H+ pumps caused hyperpolarization of the PM, which reduced the K+ loss mediated by the depolarization-activated channels in the PM of salinized roots. Therefore, we conclude that PeREM6.5 regulated H+-ATPase activity in the PM, thus enhancing the plant capacity to maintain ionic homeostasis under salinity.

Populus euphratica remorin 6.5 activates plasma membrane H+-ATPases to mediate salt tolerance.

Phytoremediation offers a great potential for affordable remediation of heavy metal (HM)-polluted soil and water. Screening and identifying candidate genes related to HM uptake and transport is prerequisite for improvement of phytoremediation by genetic engineering. Using the cadmium (Cd)-hypersensitive Populus euphratica, an annexin encoding gene facilitating Cd enrichment was identified in this study. With a 12 h exposure to CdCl (50-100 μM), P. euphratica cells down-regulated transcripts of annexin1 (PeANN1). PeANN1 was homologue to Arabidopsis annexin1 (AtANN1) and localized mainly to the plasma membrane (PM) and cytosol. Compared with wild type and Atann1 mutant, PeANN1 overexpression in Arabidopsis resulted in a more pronounced decline in survival rate and root length after a long-term Cd stress (10 d, 50 μM), due to a higher cadmium accumulation in roots. PeANN1-transgenic roots exhibited enhanced influx conductance of Cd under cadmium shock (30 min, 50 μM) and short-term stress (12 h, 50 μM). Noteworthy, the PeANN1-facilitated Cd influx was significantly inhibited by a calcium-permeable channel (CaPC) inhibitor (GdCl) but was promoted by 1 mM HO, indicating that Cd entered root cells via radical-activated CaPCs in the PM. Therefore, PeANN1 can serve as a candidate gene for improvement of phytoremediation by genetic engineering.

Populus euphratica annexin1 facilitates cadmium enrichment in transgenic Arabidopsis.

Stomatal regulation is crucial to reduce water consumption under drought conditions. Extracellular ATP (eATP) serves as a signaling agent in stomatal regulation; however, it is less known whether the eATP mediation of stomatal aperture is linked to apyrases (APYs), the principal enzymes that control the concentration of eATP. To clarify the role of APYs in stomatal control,  and  were isolated from  and transferred into Arabidopsis. Compared with the wild-type Arabidopsis and loss-of-function mutants ( and ), - and -transgenic plants decreased stomatal aperture under mannitol treatment (200 mM, 2 h) and reduced water loss during air exposure (90 min). The role of apyrase in stomatal regulation resulted from its control in eATP-regulated stomatal movements and increased stomatal sensitivity to ABA. The bi-phasic dose-responses to applied nucleotides, i.e., the low ATP (0.3-1.0 mM)-promoted opening and high ATP (>2.0 mM)-promoted closure, were both restricted by  apyrases. It is noteworthy that eATP at a low concentration (0.3 mM) counteracted ABA action in the regulation of stomatal aperture, while overexpression of  or  effectively diminished eATP promotion in opening, and consequently enhanced ABA action in closure. We postulate a speculative model of apyrase signaling in eATP- and ABA-regulated stomatal movements under drought.

 Apyrases Increase Drought Tolerance by Modulating Stomatal Aperture in Arabidopsis.

Cadmium (Cd) pollution occurring in salt-affected soils has become an increasing environmental concern in the world. Fast-growing poplars have been widely utilized for phytoremediation of soil contaminating heavy metals (HMs). However, the woody Cd-hyperaccumulator,  × , is relatively salt-sensitive and therefore cannot be directly used to remediate HMs from salt-affected soils. The aim of the present study was to testify whether colonization of  ×  with ectomycorrhizal (EM) fungi, a strategy known to enhance salt tolerance, provides an opportunity for affordable remediation of Cd-polluted saline soils. Ectomycorrhization with  strains facilitated Cd enrichment in  ×  upon CdCl exposures (50 μM, 30 min to 24 h). The fungus-stimulated Cd in roots was significantly restricted by inhibitors of plasmalemma H-ATPases and Ca-permeable channels (CaPCs), but stimulated by an activator of plasmalemma H-ATPases. NaCl (100 mM) lowered the transient and steady-state Cd influx in roots and fungal mycelia. Noteworthy,  colonization partly reverted the salt suppression of Cd uptake in poplar roots. EM fungus colonization upregulated transcription of plasmalemma H-ATPases (, , ) and annexins (, , ), which might mediate Cd conductance through CaPCs. EM roots retained relatively highly expressed  and , thus facilitating Cd enrichment under co-occurring stress of cadmium and salinity. We conclude that ectomycorrhization of woody hyperaccumulator species such as poplar could improve phytoremediation of Cd in salt-affected areas.

Ectomycorrhizal Fungal Strains Facilitate Cd Enrichment in a Woody Hyperaccumulator under Co-Existing Stress of Cadmium and Salt.

Phospholipase Dα (PLDα), which produces signaling molecules phosphatidic acid (PA), has been shown to play a critical role in plants adapting to salt environments. However, it is unclear whether phospholipase Dδ (PLDδ) can mediate the salt response in higher plants.  was isolated from salt-resistant  and transferred to  to testify the salt tolerance of transgenic plants. The NaCl treatment (130 mM) reduced the root growth and whole-plant fresh weight of wild-type (WT) , vector controls (VC) and -overexpressed lines, although a less pronounced effect was observed in transgenic plants. Under salt treatment, -transgenic Arabidopsis exhibited lower electrolyte leakage, malondialdehyde content and HO levels than WT and VC, resulting from the activated antioxidant enzymes and upregulated transcripts of genes encoding superoxide dismutase, ascorbic acid peroxidase and peroxidase. In addition, -overexpressed plants increased the transcription of genes encoding the plasma membrane Na/H antiporter () and H-ATPase (), which enabled transgenic plants to proceed with Na extrusion and reduce K loss under salinity. The capacity to regulate reactive oxygen species (ROS) and K/Na homeostasis was associated with the abundance of specific PA species in plants overexpressing . -transgenic plants retained a typically higher abundance of PA species, 34:2 (16:0-18:2), 34:3 (16:0-18:3), 36:4 (18:2-18:2), 36:5 (18:2-18:3) and 36:6 (18:3-18:3), under control and saline conditions. It is noteworthy that PA species 34:2 (16:0-18:2), 34:3 (16:0-18:3), 36:4 (18:2-18:2) and 36:5 (18:2-18:3) markedly increased in response to NaCl in transgenic plants. In conclusion, we suppose that -derived PA enhanced the salinity tolerance by regulating ROS and K/Na homeostasis in Arabidopsis.

 Phospholipase Dδ Increases Salt Tolerance by Regulating K/Na and ROS Homeostasis in Arabidopsis.

Fungal pathogens can induce canker lesions, wilting, and even dieback in many species. Trees can suffer serious physiological effects from stem cankers. In this study, we investigated the effects of  (. ) on  () leaves photosynthesis and stomatal responses, when stems were inoculated with the pathogen. To provide experimental and theoretical basis for preventing poplar canker early. One-year-old poplar stems were inoculated with .  using an epidermal scraping method. In the early stage of .  inoculation (2-14 days post inoculation, dpi), the gas exchange, stomatal dynamics, hormone content, photosynthetic pigments content, chlorophyll fluorescence parameters, and non-structural carbohydrate (NSC) were evaluated to elucidate the pathophysiological mechanism of .  inhibiting photosynthesis. Compared with the control groups, .  noteworthily inhibited the net photosynthetic rate ( ), stomatal conductance ( ), intercellular CO concentration ( ), transpiration rate ( ), and other photosynthetic parameters of poplar leaves, but stomatal limit value ( ) increased. Consistent with the above results, .  also reduced stomatal aperture and stomatal opening rate. In addition, .  not only remarkably reduced the content of photosynthetic pigments, but also decreased the maximum photochemical efficiency ( / ), actual photochemical efficiency ( ), electron transfer efficiency (ETR), and photochemical quenching coefficient ( ). Furthermore, both chlorophyll and   were positively correlated with  . In summary, the main reason for the abated   under stem canker pathogen was that .  not merely inhibited the stomatal opening, but hindered the conversion of light energy, electron transfer and light energy utilization of poplar leaves. In general, the lessened CO and   would reduce the synthesis of photosynthetic products. Whereas, sucrose and starch accumulated in poplar leaves, which may be due to the local damage caused by .  inoculation in phloem, hindering downward transport of these products.

Stem canker pathogen  inhibits poplar leaf photosynthesis in the early stage of inoculation.

A key step in the study of tree pathology is the identification of an appropriate method for inoculating pathogens of diseases in branches and trunks. Pathogens of diseases in branches and trunks are commonly inoculated through punching, burning, and toothpick inoculation. However, there is a lack of comparative analyses of the inoculation outcomes of these three methods. In this work, six-year-old  var.  were inoculated with  using punching, burning, and toothpick techniques to investigate the differences in the effectiveness of these inoculation methods. Results reveal that the incidence rate was 93.55% in the toothpick inoculation group, significantly higher than the 80.65% in the burning inoculation group (chi-square,  = 90,  = 0.007), while punching inoculation exhibited significant pathological responses in the early stages, with spontaneous healing in the later stage. Additionally, toothpick inoculation was more efficient in inducing  canker when inoculating the pathogen at the bottom of the tree, with lower intra- and inter-row spacing (stand density) providing better outcomes than higher intra- and inter-row spacing. The results of this study demonstrate that toothpick inoculation is an optimal option for studying the artificial inoculation of  in six-year-old  var. , providing technical support for research on poplar diseases and offering a theoretical basis for the inoculation of other diseases in the branch and trunk.

Article	Year
Biology and behavior of Spathius agrili, a parasitoid of the emerald ash borer, Agrilus planipennis, in China. Journal of insect science (Online)\| PubMed ID: 20569125	2010
The biology and ecology of the emerald ash borer, Agrilus planipennis, in China. Journal of insect science (Online)\| PubMed ID: 20879922	2010
Salt-Sensitive Signaling Networks in the Mediation of K/Na Homeostasis Gene Expression in Roots. Frontiers in plant science\| PubMed ID: 28855912	2017
The Arabidopsis Ca-Dependent Protein Kinase CPK12 Is Involved in Plant Response to Salt Stress. International journal of molecular sciences\| PubMed ID: 30558245	2018
JRL Mediates ABA Response, Ionic and ROS Homeostasis in Arabidopsis under Salt Stress. International journal of molecular sciences\| PubMed ID: 30769802	2019
Fungal canker pathogens trigger carbon starvation by inhibiting carbon metabolism in poplar stems. Scientific reports\| PubMed ID: 31300723	2019
Populus euphratica WRKY1 binds the promoter of H+-ATPase gene to enhance gene expression and salt tolerance. Journal of experimental botany\| PubMed ID: 31680166	2020
Populus euphratica remorin 6.5 activates plasma membrane H+-ATPases to mediate salt tolerance. Tree physiology\| PubMed ID: 32159803	2020
Populus euphratica annexin1 facilitates cadmium enrichment in transgenic Arabidopsis. Journal of hazardous materials\| PubMed ID: 33092878	2021
Apyrases Increase Drought Tolerance by Modulating Stomatal Aperture in Arabidopsis. International journal of molecular sciences\| PubMed ID: 34576057	2021
Ectomycorrhizal Fungal Strains Facilitate Cd Enrichment in a Woody Hyperaccumulator under Co-Existing Stress of Cadmium and Salt. International journal of molecular sciences\| PubMed ID: 34769083	2021
Phospholipase Dδ Increases Salt Tolerance by Regulating K/Na and ROS Homeostasis in Arabidopsis. International journal of molecular sciences\| PubMed ID: 35563299	2022
Stem canker pathogen inhibits poplar leaf photosynthesis in the early stage of inoculation. Frontiers in plant science\| PubMed ID: 36204063	2022
Comparative Study on the Effectiveness of Three Inoculation Methods for in var. . Biology\| PubMed ID: 38666863	2024

Yinan Zhang

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