Molecular,Biological and Phylogenetic Analysis of Chinese Isolates of Hop stunt viroid Associated with Citrus Cachexia Disease

Citrus cachexia is an economically important disease of citrus hosts caused by specific variants of Hop stunt viroid (HSVd) that are usually referred to as Citrus cachexia viroid (CCaVd). Eight cachexia‐associated HSVd isolates were collected from six citrus growing areas of China, where citrus cachexia had not been reported previously. Forty‐seven independent cDNA clones were used for genetic diversity and phylogenetic analysis. There were no sequence variant‐cultivar correlation and no distinct regional specificity among or within the cachexia‐associated HSVd populations analysed. Three clusters consisting of three major HSVd variants were identified by phylogenetic analysis, suggesting that most Chinese isolates contain a mixture of cachexia and non‐cachexia variants. Biological properties of eight CCaVd isolates were determined by inoculating Parson’s Special mandarin (Citrus reticulata). Our results suggest that the interactions between CCaVd and non‐CCaVd variants might play an important role in suppressing cachexia symptom expression.     

4-Desmethylsterol composition of citrus rootstocks of different salt exclusion capacity

Fibrous roots from seedlings of three citrus rootstocks (Rungpur lime, Kharna khatta and Etrog citron) grown hydroponically for 6 weeks in the presence or absence of 50 mM NaCl were analysed for their content of free, esterified, and glycosidic sterols. Leaf chloride analyses indicated that Rangpur lime was a good Cl- excluder and the other two rootstocks were Cl- accumulators.
On a dry weight basis and in the absence of NaCI only the free 4-desmethyIsterol levels showed significant rootstock differences. Kharna khatta had the highest, and Rangpur lime the lowest, free stcrol levels. Sitostcrol was the major component of all sterol fractions of Rangpur lime, the esterified fraction of the other rootstocks, and the glycosidic sterol fraction of Kharna khatta. The ratio of sitosterol to stigmasterol was highest in Rangpur lime and lowest in Etrog citron in all cases and the ratio of apos;more-planar apos; to apos;less-planar apos; free sterols was highest in Etrog citron and lowest in Rangpur lime.
Treatment with 50 mM NaCI resulted in an increase in free sterol levels in Rangpur lime and a decrease in Kharna khatta. Steryl ester levels were unaffected in Rangpur lime but were significantly reduced in the other rootstocks. In all three sterol fractions the sitosterol/stigmasterol ratio was decreased. A decrease in the ratio of apos;more-planarapos; to apos;less-planarapos; free sterols was observed only in Kharna khatta and, more notably, Etrog citron. Salt-induced changes in the apos;more-planar apos; to apos;less-planar apos; free sterol ratio correlated well with salt exclusion capacity.     

Interference between viroids inducing exocortis and cachexia diseases of citrus

Systemic interactions of long duration among the Group I] Citrus Viroids, CVd-IIa and CVd-IIb were investigated by grafting buds from established citron (Citrus medica L.) sources containing single viroids to a common healthy seedling. In healthy tissues, the two viroids became established in approximately equal titres as a mixed infection. By contrast, tissues that grew from the CVd-IIa or CVd-IIb source buds contained only trace amounts of the heterologous invading viroids. This interference between CVd-IIa, a mild exocortis agent, and CVd-IIb, the cachexia agent, was also demonstrated in the presence of CEVd, the severe exocortis agent but not a Group II viroid. When a CVd-IIb bud was propagated on a citron containing CVd-IIa, a dramatic reduction in the titre of CVd-IIb was observed. The interference between CVd-IIa and CVd-IIb indicates that the mild and relatively innocuous isolate, CVd-IIa, can interfere with the replication and/or accumulation of the severe cachexia agent, CVd-IIb, in citrus. This offers a potential practical approach for the control of cachexia in commercial plantings by “viroid interference”.     

An artificial chimeric derivative of Citrus viroid V involves the terminal left domain in pathogenicity

The recently described Citrus viroid V (CVd-V) induces, in Etrog citron, mild stunting and very small necrotic lesions and cracks, sometimes filled with gum. As Etrog citron plants co-infected with Citrus dwarfing viroid (CDVd) and CVd-V show synergistic interactions, these host–viroid combinations provide a convenient model to identify the pathogenicity determinant(s). The biological effects of replacing limited portions of the rod-like structure of CVd-V with the corresponding portions of CDVd are reported. Chimeric constructs were synthesized using a novel polymerase chain reaction-based approach, much more flexible than those based on restriction enzymes used in previous studies. Of the seven chimeras (Ch) tested, only one (Ch5) proved to be infectious. Plants infected with Ch5 showed no symptoms and, although this novel chimera was able to replicate to relatively high titres in singly infected plants, it was rapidly displaced by either CVd-V or CDVd in doubly infected plants. The results demonstrate that direct interaction(s) between structural elements in the viroid RNA (in this case, the terminal left domain) and as yet unidentified host factors play an important role in modulating viroid pathogenicity. This is the first pathogenic determinant mapped in species of the genus Apscaviroid .     

Phospholipids, free sterols and adenosine triphosphatase of plasma membrane-enriched preparations from roots of citrus genotypes differing in chloride exclusion ability

Plasma membrane-enriched preparations from fibrous roots of three citrus genotypes differing in their abilities for chloride exclusion, and grown in the presence of 0,50 or 100 mM NaCl for 4 weeks, were analysed for phospholipid and free sterol content and vanadate-sensitive adenosine triphosphatase (ATPase) activity over a range of temperatures. The best chloride excluder, Rangpur lime (Citrus reticulaia Blanco var. austera hyb.?), had significantly higher maximal ATPase activity than both the moderate chloride excluder. Kharna khatta (Citrus kharna Raf.), and the worst chloride excluder, Etrog citron (Citrus medico L.), at all assay temperatures below 28°C. Salt treatment had no effect on maximal ATPase activity of either Rangpur lime or Etrog citron but resulted in increased activity of the enzyme in Kharna khatta at temperatures below 28°C. Arrhenius plots of ATPase activity from the three citrus genotypes showed that, in controls, the activation energy (E.,) of Rangpur lime ATPase was significantly lower than that of both Kharna khatta and Etrog citron. The thermotropic phase transition temperature (T_f) for Rangpur lime (27°C) was also lower than for the other citrus genotypes (31°C). Salt treatment resulted in increases in both Ea and T, for Rangpur lime, decreases in both parameters for Kharna khatta and no change of either parameter for Etrog citron. An inverse relationship between E_a and the phospholipid to free sterol ratio was evident for plasma membrane preparations from all three citrus genotypes in the presence and absence of salt treatment suggesting that changes in membrane fluidity, particularly those induced by free sterols, have the potential to influence active as well as passive ion transport processes and thus may play a significant role in the chloride exclusion mechanism.     

Characterization of Two Fungal Isolates from Cotton and Evaluation of their Potential for Biocontrol of Verticillium Wilt of Cotton

Two isolates (CVd‐WHw and CVn‐WHg) recovered from Verticillium‐wilt‐symptomatic cotton grown in Hubei Province of China were identified based on their morphology, growth characteristics in culture, specific amplification and identification of internal transcribed spacer (ITS) rDNA sequence. According to the morphological characteristics, specific PCR amplification and ITS sequences, CVd‐WHw was identified as V. dahliae and CVn‐WHg as Gibellulopsis nigrescens. In bioassays, the two isolates had significantly lower pathogenicity to cotton plant than V. dahliae isolate CVd‐AYb. Cotton pre‐inoculated with isolate CVn‐WHg or CVd‐WHw exhibited reduced disease indices of Verticillium wilt compared with those inoculated with CVd‐AYb alone. Cotton co‐inoculated with CVn‐WHg or CVd‐WHw and CVd‐AYb provided increased protection from subsequent CVd‐AYb inoculation. These results suggest that the two isolates have the potential to be developed as biocontrol agents for the control of Verticillium wilt in cotton. To our knowledge, this is the first report of a cross‐protection phenomenon using Gibellulopsis nigrescens against Verticillium wilt caused by V. dahliae on cotton.     

NaCl effects on 4-desmethylsterol composition of plasma-membrane-enriched preparations from citrus roots

Abstract The free 4-desmethylsterol composition of plasma-membrane-enriched preparations from white fibrous roots of Rangpur lime (Citrus reticulata var. austera hybrid?), Kharna khatta (C. kharna Raf.) and Etrog citron (C. medica L.) seedlings grown in the presence of 0, 50, or 100 mol m^?3 NaCl for 28 d was quantitated by gas chromatography (GC) on analytical capillary (SE-54 fused silica) columns and the sterols were identified by combined gas chromatography-mass spectrometry (GC-MS). Only three 4-desmethylsterols were positively identified by GC-MS, viz. campesterol, stigmasterol and sitosterol. Cholesterol could not be positively identified in any of the membrane preparations. Campesterol levels were generally similar for all treatments and for all three genotypes, approximating 30% of the total free 4-desmethylsterol content of the plasma membranes. At all levels of salinity (0, 50 or 100 mol m^?3 NaCl) sitosterol levels decreased in the order Rangpur lime > Kharna khatta > Etrog citron and stigmasterol levels decreased in the reverse order. The ratio of sitosterol to stigmasterol was highest in Rangpur lime and lowest in Etrog citron at each level of salinity and was reduced by salt treatment in all three genotypes. Salt-induced reductions in the ratio of ‘more planar’ to ‘less planar’ sterols correlated inversely with the accumulation of Cl^? in the leaves of the three genotypes suggesting a role for plasma membrane sterols in the Cl^? exclusion mechanism. A model relating sterol structure, membrane sterol composition and membrane permeability to Cl^? exclusion ability in citrus is presented.     

Studies on Taxonomy and Evolution of Citrus and Its Related Genera by Isozyme Analysis

The leaf or bark tissue extracts of 108 biotypes of Citrus and its five related genera were analyzed by polyacrylamide gel electrophoresis for isozymes of peroxidase, glutamate oxaloacetate transaminase, phosphoglucose isomerase, phosphoglucose mutase, superoxide dismutase, tetrazolium oxidase, NADP+- dependent malate dehydrogenase and esterase. The inter-or intra-generic differences of isozymograms were compared. Dissimilarities of the isozyme patterns among the samples were calculated by computer based on the UPGMA method to demonstrate the phylogenetic relationship of the biotypes. There are remarkable isozymogramatic differences among the six genera. Generally speaking, each genus possesses its own unique bands. Based on the cluster analysis, the total Citrus biotypes are classified into seven groups, i.e. Honghe papeda, Mauritius papeda, Pummelo, Ichang papeda, Citron, Lemon-lime and Mandarin-orange. Ichang papeda is suggested to be the third subgenus of the genus Citrus. The Fuming trifoliata orange is accepted as a new species of Poncirus-P. polyandra S. Q. Ding. It might be the evolutionary bridge between Poncirus and Citrus, and one of the “missing links” as suggested by W. T. Swingle. Microacrumen (small- fruited mandarin) is the primitive form of the mandarins. It seems better to consid er“ Horse-nose mandarin ”or “Xipigoushigan mandarin” as the typic mandarin originated in China. Euacrumen (large-fruited mandarin) might have arised through two ways. One is hybridization of Microacrumen and C. sinensis, and the other is just the variation of Microacrumen. Ancestors of some biotypes are inferred based on isozyme data. Fortunella obovata Tan. may be a hybrid of Fortunella and Citrus. C. unshiu Marc. arose from the nucellar mutation of“Huangyanbendiguangju mandarin”( C. nobilis Lour.) in Huangyan of Zhejiang Province. C. junos Sieb. came from hybridization of Ichang papeda and mandarin. C. grandis Osbeck and mandarin may have been involved in the origin of C. aurantium L.. C. limonia Osbeck may be a hybrid between C. medica L. and mandarin. The evolutionary trend of citrus fruit trees is proposed, anda tentative phylogenetic diagram is drawn.     

Microarray analysis of Etrog citron (Citrus medica L.) reveals changes in chloroplast, cell wall, peroxidase and symporter activities in response to viroid infection

Viroids are small (246-401 nucleotides), single-stranded, circular RNA molecules that infect several crop plants and can cause diseases of economic importance. Citrus are the hosts in which the largest number of viroids have been identified. Citrus exocortis viroid (CEVd), the causal agent of citrus exocortis disease, induces considerable losses in citrus crops. Changes in the gene expression profile during the early (pre-symptomatic) and late (post-symptomatic) stages of Etrog citron infected with CEVd were investigated using a citrus cDNA microarray. MaSigPro analysis was performed and, on the basis of gene expression profiles as a function of the time after infection, the differentially expressed genes were classified into five clusters. FatiScan analysis revealed significant enrichment of functional categories for each cluster, indicating that viroid infection triggers important changes in chloroplast, cell wall, peroxidase and symporter activities.     

Tree‐sequence recording in SLiM opens new horizons for forward‐time simulation of whole genomes

There is an increasing demand for evolutionary models to incorporate relatively realistic dynamics, ranging from selection at many genomic sites to complex demography, population structure, and ecological interactions. Such models can generally be implemented as individual‐based forward simulations, but the large computational overhead of these models often makes simulation of whole chromosome sequences in large populations infeasible. This situation presents an important obstacle to the field that requires conceptual advances to overcome. The recently developed tree‐sequence recording method (Kelleher, Thornton, Ashander, & Ralph, 2018), which stores the genealogical history of all genomes in the simulated population, could provide such an advance. This method has several benefits: (1) it allows neutral mutations to be omitted entirely from forward‐time simulations and added later, thereby dramatically improving computational efficiency; (2) it allows neutral burn‐in to be constructed extremely efficiently after the fact, using “recapitation”; (3) it allows direct examination and analysis of the genealogical trees along the genome; and (4) it provides a compact representation of a population's genealogy that can be analysed in Python using the msprime package. We have implemented the tree‐sequence recording method in SLiM 3 (a free, open‐source evolutionary simulation software package) and extended it to allow the recording of non‐neutral mutations, greatly broadening the utility of this method. To demonstrate the versatility and performance of this approach, we showcase several practical applications that would have been beyond the reach of previously existing methods, opening up new horizons for the modelling and exploration of evolutionary processes.     

Two nucleotide positions in the Citrus exocortis viroid RNA associated with symptom expression in Etrog citron but not in experimental herbaceous hosts

Citrus exocortis viroid (CEVd) is the causal agent of exocortis disease of citrus. CEVd has a wide host range that includes woody and herbaceous species. A new CEVd strain (CEVd(COL)), phylogenetically clustering with CEVd variants of Class A inducing severe symptoms in tomato, was identified in Colombia and shown to induce only extremely mild symptoms in Etrog citron indicator plants. Using site-directed mutagenesis, two nucleotide substitutions (314A → G and 315U → A) in the lower strand of the P domain of the predicted CEVd(COL) secondary structure resulted in a severe artificial CEVd(MCOL) variant. Conversely, two nucleotide exchanges (314G → A and 315A → U) in the same region of the severe variant CEVd(E-117) resulted in a symptomless artificial CEVd(ME-117) variant. Infectivity assays conducted with the natural and mutated variants showed that all induced severe symptoms in Gynura aurantiaca, tomato and chrysanthemum. This is the first report of the identification of pathogenic determinants of CEVd in citrus, and shows that these pathogenicity determinants are host dependent.     

Differential Responses of the Whitefly Bemisia tabaci Symbionts to Unfavorable Low and High Temperatures

The whitefly Bemisia tabaci complex contains many cryptic species, of which the Middle East-Asia Minor 1 (MEAM1) and Mediterranean (MED) are notorious invasive pests. In our field-collected whitefly samples, MEAM1 harbors an obligate primary symbiont “Candidatus Portiera aleyrodidarum” and two secondary symbionts, “Candidatus Hamiltonella defensa” and Rickettsia sp., whereas MED has only “Ca. Portiera aleyrodidarum” and “Ca. Hamiltonella defensa.” Both “Ca. Portiera aleyrodidarum” and “Ca. Hamiltonella defensa” are intracellular endosymbionts residing in the bacteriomes, whereas Rickettsia sp. has a scattered distribution throughout the host body cavity. We examined responses of these symbionts to adverse temperatures as well as survival of the host insects. After cold treatment at 5 or 10 °C or heat treatment at 35 or 40 °C for 24 h, respectively, the infection rates of all symbionts were not significantly decreased based on diagnosis PCR. However, quantitative PCR assays indicated significant reduction of “Ca. Hamiltonella defensa” at 40 °C, and the reduction became greater as the duration increased. Compared with “Ca. Hamiltonella defensa,” “Ca. Portiera aleyrodidarum” was initially less affected in the first day but then showed more rapid reduction at days 3–5. The density of Rickettsia sp. fluctuated but was not reduced significantly at 40 °C. Meanwhile, the mortality rates of the host whiteflies elevated rapidly as the duration of exposure to heat treatment increased. The differential responses of various symbionts to adverse temperatures imply complex interactions among the symbionts inside the same host insect and highlight the importance of taking the whole bacterial community into account in studies of symbioses.     

Some Effects of Root Anatomy on K, Na and Cl Loading of Citrus Roots and Leaves

Loading of K, Na and Cl into fibrous roots of salt-treated citrusgenotypes, Rangpur lime (Citrus reticulata var. austera hybrid?)and Etrog citron (C. medica L.) was investigated in relationto root anatomy, in particular, the differentiation of the epidermal-hypodermallayers with distance from the root tip. The influence of durationof salinity treatment on the characteristics of K, Na and Claccumulation in leaves of the two genotypes was explored intwo experiments respectively, covering the short term (14 d)and long term (12 weeks). This study focused on two regions of the fibrous root, a segment2–12 mm from the root tip, immediately basipetal to thezone of elongation and a differentiated region of the maturesuberized, fibrous root, 40–50 mm from the root tip. Inthe distal root segment (2–12 mm) the epidermis and hypodermisof both genotypes was observed as two closely packed, uniseriatelayers of living cells. In the proximal root segment (40–50mm) the differentiated hypodermis was evident as a uniseriatelayer of thick-walled lumina interspersed with ‘passagecells’ which were frequently associated with clustersof viable epidermal cells. The characteristics of Na and Cl loading in the two root zonesdiffered profoundly during the short term loading (acclimation)phase. Attainment of quasi-steady-states for Na and Cl in thedistal region (with the exception of Na in Etrog citron) wasrapid as was Na equilibration of the proximal root segmentsin both genotypes. In contrast, Cl loading in the proximal regiontook c. 14 d to reach a quasi-steady-state by which time Cllevels were 2 to 3 times higher in the proximal than in thedistal root segments. The superior tolerance of Rangpur lime to long term salinitywas highly correlated to Cl exclusion from the leaves. However,during the first 14 d of acclimation to 50 mol m^–3 NaClthere was no segregation of the two genotypes based upon leafCl levels. Expression of differential accumulation of Cl inleaves appeared to be a time dependent process and was manifestonly after Cl saturation of the proximal root which representsthe bulk of the fibrous root system. The salt tolerance of Rangpurwas also associated with high selectivity of fibrous roots forK. over Na. A pronounced loss of K from cortical cells in theproximal root segment of salt-stressed Etrog citron was alsoevident by X-ray microanalysis. Key words: Citrus, anatomy, salinity, roots, X-ray microanalysis     

Autoimmunity and the microbiome: T‐cell receptor mimicry of “self” and microbial antigens mediates self tolerance in holobionts

I propose a T‐cell receptor (TcR)‐based mechanism by which immunity mediates both “genetic self” and “microbial self” thereby, connecting microbiome disease with autoimmunity. The hypothesis is based on simple principles. First, TcR are selected to avoid strong cross‐reactivity with “self,” resulting in selection for a TcR repertoire mimicking “genetic self.” Second, evolution has selected for a “microbial self” that mimics “genetic self” so as to share tolerance. In consequence, our TcR repertoire also mimics microbiome antigenicity, providing a novel mechanism for modulating tolerance to it. Also, the microbiome mimics the TcR repertoire, acting as a secondary immune system. I call this TcR‐microbiome mimicry “holoimmunity” to denote immune tolerance to the “holobiont self.” Logically, microbiome‐host mimicry means that autoimmunity directed at host antigens will also attack components of the microbiome, and conversely, an immunological attack on the microbiome may cross‐react with host antigens producing “holoautoimmunity.”

     

Susceptibility and Resistance of Citrus Genotypes to Alternaria alternata pv. citri

A survey of citrus cultivars in Israel in orchards where Alternaria brown spot was common on Minneola tangelos (mandarin × grapefruit), revealed the occurrence of the disease as typical foliar and fruit lesions on Dancy and Ellendale (mandarins), on Murcott tangor (mandarin × sweet orange), on Nova and Idith (mandarin hybrids), on Calamondin, and on Sunrise and Redblush (grapefruit). Isolates of Alternaria alternata from each of these hosts were proven to be pathogenic to Minneola tangelo.
The host range of A. alternata pv. citri from Israel was assayed by inoculating leaves of diverse citrus genotypes. Several mandarins and their hybrids (Dancy, Kara, King, Wilking, Satsuma, Minneola, Orlando, Mikhal, Idith, Nova, Page, Murcott), grapefruit (Marsh seedless), grapefruit × pummelo (Oroblanco), sweet orange (Shamouti, Valencia, Washington navel) Calamondin, and Volkamer citrus were susceptible. Several mandarins and their hybrids (Clementine, Avana, Yafit, Ortanique), Cleopatra, one sweet orange cultivar (Newhall), pummelo (Chandler), lemon (Eureka), Rough lemon, Rangpur lime, sweet lime, citron, limequat, sour orange, Troyer citrange and Alemow were resistant.     

Interactive effects between diet and genotypes of host and pathogen define the severity of infection

Host resistance and parasite virulence are influenced by multiple interacting factors in complex natural communities. Yet, these interactive effects are seldom studied concurrently, resulting in poor understanding of host‐pathogen‐environment dynamics. Here, we investigated how the level of opportunist pathogen virulence, strength of host immunity and the host condition manipulated via diet affect the survival of wood tiger moth Parasemia plantaginis (Arctidae). Larvae from “low cuticular melanin” and “high cuticular melanin” (considered as low and high pathogen resistance, respectively) selection lines were infected with moderately and highly virulent bacteria strains of Serratia marcescens, while simultaneously manipulating host diet (with or without antibacterial compounds). We measured host survival and food preference before and after infection to test whether the larvae “self‐medicate” by choosing an anti‐infection diet (Plantago major, i.e., plantain leaf) over lettuce (Lactuca sativa). “High melanin” larvae were more resistant than “low melanin” larvae to the less virulent strain that had slower growth and colonization rate compared with the more virulent strain. Cuticular melanin did not enhance survival when the larvae were infected with the highly virulent strain. Anti‐infection diet enhanced survival of the “high melanin” but not the “low melanin” hosts. Survival was dependent on family origin even within the melanin selection lines. Despite the intrinsic preference for lettuce, no evidence of self‐medication was found. These results demonstrate that the relative benefit of host cuticular melanin depends on both diet and pathogen virulence: plantain diet only boosted the immunity of already resistant “high melanin” hosts, and cuticular melanin increased host survival only when infected with moderately virulent pathogen. Moreover, there was considerable variation in host survival between families within both melanin lines suggesting genetic basis for resistance. These results indicate that although melanin is an important predictor of insect immunity, its effect on disease outcomes greatly depends on other interacting factors.     

Partial nephrectomy margin imaging using structured illumination microscopy

Partial nephrectomy (PN) is the recommended procedure over radical nephrectomy (RN) for patients with renal masses less than 4 cm in diameter (Stage T1a). Patients with less than 4 cm renal masses can also be treated with PN, but have a higher risk for positive surgical margins (PSM). PSM, when present, are indicative of poor clinical outcomes. The current gold‐standard histopathology method is not well‐suited for the identification of PSM intraoperatively due to processing time and destructive nature. Here, video‐rate structured illumination microscopy (VR‐SIM) was investigated as a potential tool for PSM detection during PN. A clinical image atlas assembled from ex vivo renal biopsies provided diagnostically useful images of benign and malignant kidney, similar to permanent histopathology. VR‐SIM was then used to image entire parenchymal margins of tumor resection covering up to >1800× more margin surface area than standard histology. Aided by the image atlas, the study pathologist correctly classified all parenchymal margins as negative for PSM with VR‐SIM, compared to standard postoperative pathology. The ability to evaluate large surgical margins in a short time frame with VR‐SIM may allow it to be used intraoperatively as a “safety net” for PSM detection, allowing more patients to undergo PN over RN.