Nucleotide Sequence of Citrus Tristeza Virus Seedling Yellows Isolate

The complete nucleotide sequence of a seedling-yellows-inducing isolate NUagA of Citrus tristeza virus (CTV) was determined. It consisted of 19302 nucleotides and contained 12 open reading frames (ORF) organized identically to those of previously sequenced isolates. This genome is the largest among the CTV genome sequenced so far ; it is 6 nucleotides (nt), 76 nt, 43 nt, and 53 nt longer than that of T36 (quick decline, Florida), VT (seedling yellows, Israel), T385 (mild, Spain), and SY568 (stem pitting, California), respectively. Sequence comparison of NUagA and the other isolates revealed approximately 90% identities throughout the 3′ half of the genome. The 5′ half of the genome was only about 70% identical to that of T36 but still high at about 90% to those of VT, SY568, and T385. Comparison of amino acid sequences on ORF1a encoding polyproteins, the most variable region, reflects the CTV isolate relationship ; NUagA is closely related to VT, SY568, and T385, but distantly related to T36. Received 29 May 2000/ Accepted in revised form 16 November 2000     

Selective transmission of the seedling yellows-type genotypic variants of Citrus tristeza virus by Aphis gossypii in northern Iran

Citrus tristeza virus (CTV) has existed in northern Iran for more than five decades. The long-time interaction of different virus genotypes with Aphis gossypii, as the only aphid vector of CTV in northern Iran, has led to the emergence of highly virulent subpopulations, among others, in the established foci. Here, we studied the population structure of the originally established CTV isolates present in Satsuma mandarin (Citrus unshiu) trees imported from Japan, and subisolates thereof, formed following experimental transmission by A. gossypii, as well as those evolved through natural transmission by this aphid species in the groves. Symptoms of the naturally spread and the experimentally aphid-transmitted isolates were similar to those of the Satsuma CTV source isolates for all indicator plants except for sour orange (Citrus aurantium) and grapefruit (Citrus paradisi), with the aphid-transmitted isolates additionally inducing severe seedling yellows and stunting in these two indicators. Studies on the population heterogeneity of these isolates through comparison of their single-strand conformational polymorphism profiles and nucleotide sequences of the 25 kDa capsid protein gene from the predominant haplotypes, and dot-blot hybridization signals, revealed the presence of two major T36- and SY568- (or NUagA-) like genotypes along with a minor poorly characterized one in the originally infected Satsuma trees; in contrast, only a certain genomic variant having the highest similarity to the isolate SY568 (and NUagA) was predominant both in the naturally infected trees and in those infected experimentally by A. gossypii. It seems that transmission by A. gossypii to sweet orange (Citrus sinensis) has led to the preponderance of the CTV genomic variants inducing severe seedling yellows in northern Iran.     

Characterization of Citrus tristeza virus isolates in northern Iran

The biological and molecular properties of four Citrus tristeza virus (CTV) isolates isolated from infected Satsuma trees imported from Japan, and growing in citrus groves in northern Iran (Mahdasht orchards, Mazandaran Province), were investigated. CTV-infected samples were collected from sweet orange trees and grafted onto Alemow (Citrus macrophylla Wester) seedlings. On indicator plants, these isolates produced various symptoms including vein clearing and stem pitting on Mexican lime, Alemow, and Citrus hystrix, and yellowing and stunting on sour orange and grapefruit seedlings. Citrus samples were also surveyed for CTV using serological tests. The coat protein (CP) gene of these isolates was amplified using specific primers, yielding an amplicon of 672 bp for all isolates. Sequence analysis showed 98%–99% sequence homology of Iranian isolates with the Californian CTV severe stem-pitting isolate SY568 and 97%–98% homology with the Japanese seedling yellows isolate NUagA. The Iranian isolates were compared by restriction fragment length polymorphism (RFLP) analysis of the CP amplicon for further classification.     

First detection of a virulent strain of Citrus tristeza virus (Closteroviridae) in a citrus orchard of Chlef Valley (Algeria)

A large‐scale survey of Citrus tristeza virus (CTV) was carried out from 2016 to 2018 in the Chlef Valley, one of the main citrus growing areas in Algeria. In this study a total of 1680 citrus trees from 93 commercial orchards were sampled. The collected samples were tested by direct tissue blot immunoassay analysis and by the double antibody sandwich enzyme‐linked immunosorbent assay technique, and 54 trees were identified as being infected with CTV. This result confirmed that 54 trees were infected by the virus, corresponding to an infection rate of 3.21% throughout the studied area. Five of these local CTV sources were chosen for further molecular investigations to determine the genotype associated with the CTV isolates now spreading in the Chlef area. Characterization with multiple molecular markers showed the presence of the T30 and VT genotypes. This result allowed confirmation of the presence of a virulent strain belonging to the VT genotype. The other CTV isolates were similar to those from the Mitidja region, which showed 99% nucleotide identity with the Spanish mild CTV isolate. This early finding of a strain belonging to the VT genotype is an issue for Algerian citrus producers and needs rapid actions to be taken by the National phytosanitary services, extending the surveillance to other citrus production regions and uprooting the infected trees.     

Biological, serological and molecular characterization of three isolates of Citrus tristeza closterovirus introduced into Morocco

The biological, serological and genomic diversity of three Citrus tristeza virus (CTV) isolates from various geographical regions was studied: isolate P1 from lemon cv. 'Meyer' in a field near Marrakech (MA) in 1983, and isolates P2 and R1 detected in imported Spanish clementine germplasm by the Moroccan NPPO in 1998 and 2000. P1 induced severe vein clearing on Mexican lime and grapefruit, mild stem pitting on Mexican lime and moderate stem pitting on grapefruit. P2 and R1 only induced mild vein clearing on Mexican lime and caused no stem pitting or other symptoms on indicator plants used as controls. Only isolate P1 reacted with monoclonal antibody MCA-13, whereas all isolates reacted positively with the 3DF1+3CA5 mixture. The Moroccan clones P1–3 and P1–5, and all other severe isolates obtained from GenBank, showed a phenylalanine at amino acid position 124 of their coat protein sequences. This epitope confers MCA13 reactivity. The Spanish clones had tyrosine instead at this position. The deduced amino-acid sequence of coat protein of P1 clones clusters close to severe strains CB3–104 and FL7, respectively from Brazil and USA (Florida) (Group 5), whereas the sequences from P2 and R1 cluster close to typical strains from Portugal 25–120 and USA (Florida) T30 (Group M). The three techniques for distinguishing CTV isolates were clearly correlated.     

Genetic Marker Analysis of a Global Collection of Isolates of Citrus tristeza virus: Characterization and Distribution of CTV Genotypes and Association with Symptoms

ABSTRACT Genetic markers amplified from three noncontiguous regions by sequence specific primers designed from the partial or complete genome sequences of Citrus tristeza virus (CTV) isolates T3, T30, T36, and VT were used to assess genetic relatedness of 372 isolates in an international collection. Eighty-five isolates were judged similar to the T3 isolate, 81 to T30, 11 to T36, and 89 to VT. Fifty-one isolates were mixed infections by two or more identifiable viral genotypes, and 55 isolates could not be assigned unequivocally to a group defined by marker patterns. Maximum parsimony analysis of aligned marker sequences supported the grouping of isolates on the basis of marker patterns only. Specific disease symptoms induced in select citrus host plants were shared across molecular groups, although symptoms were least severe among isolates grouped by markers with the T30 isolate and were most severe among isolates grouped by markers with the T3 isolate. Isolates assigned the same genotype showed variable symptoms and symptom severity. A classification strategy for CTV isolates is proposed that combines genetic marker patterns and nucleotide sequence data.     

Medicago truncatula as a model host for studying legume infecting Rhizoctonia solani and identification of a locus affecting resistance to root canker

The broad‐host‐range necrotizing fungal pathogen Rhizoctonia solani is responsible for economically significant diseases to crops as diverse as wheat, maize, barley, canola, sugar beet, potato, soyabean, bean, lupin and alfalfa. Germplasm screens in many of the crop hosts have not identified strong genetic resistance which, together with the lack of effective control, mean the pathogen remains a substantial problem for agriculture in many parts of the world. Following the establishment of a robust inoculation assay, a germplasm collection of the model legume Medicago truncatula was screened with various legume‐infecting isolates of R. solani. While some significant differences in susceptibility/resistance were detected between some lines, in the majority of cases M. truncatula was susceptible to R. solani. Comparison of a legume‐ and cereal‐infecting AG8 isolate with a legume‐specific AG11 isolate revealed no difference in pathogenicity between the two isolates when infecting M. truncatula. The most significant differences in susceptibility occurred with an AG6 isolate, which caused root canker. This included significant differences between the moderate resistance of the M. truncatula reference genotype A17 and the high susceptibility of line A20. The analysis of a recombinant inbred line population derived from A17 and A20 revealed a single locus contributing to the resistance in A17. Interestingly, the locus only affected the development of post‐emergent (late) symptoms, such as necrosis of cotyledons at 11 days after inoculation and root‐ and above‐ground‐weights, but not pre‐emergent seedling damping off. These findings pave the way for further studies to dissect the genetic and molecular mechanisms of resistance.     

Fusarium solani f. sp. passiflorae: a new forma specialis causing collar rot in yellow passion fruit

The aim of this study was to characterize a Fusarium population obtained from yellow passion fruit (YPF) with collar rot using pathogenicity, morphocultural characteristics and molecular tests. Pathogenicity and disease severity were assessed in six plant species: YPF, zucchini, tomato, bean, soya bean and cucumber. Potato dextrose agar medium (PDA) was used to determine mycelial growth at five temperatures (15–35°C). The colour produced by isolates was also determined on PDA at 25°C. Synthetic nutrient agar medium was used to evaluate: (i) type of mycelium and phialides; (ii) size, shape and number of septa from conidia; and (iii) production of chlamydospores and perithecia. Molecular tests consisted of sequencing the ITS–5·8S rDNA region and elongation factor 1α (EF‐1α) gene. The isolates caused large lesions on YPF, zucchini and tomato, with YPF having the highest mean disease severity and being the only one that showed wilt symptoms and death of the plant. Thus the isolates showed host specificity. Maximum mycelial growth occurred at 25°C and the predominant colour was bluish‐white. The isolates produced long phialides, dense aerial mycelium, oval microconidia with a mean size of 9·5 × 2·6 μm, macroconidia of 32·7 × 3·4 μm with 3·3 septa, and chlamydospores; only one isolate lacked perithecia. Phylogenetic trees of the ITS region and EF‐1α gene showed that isolates from YPF formed a distinct group within the F. solani group and the formae speciales of F. solani. It is proposed to name all isolates from YPF as F. solani f. sp. passiflorae.     

Identification of isolate‐specific resistance QTLs to phytophthora root rot using an intraspecific recombinant inbred line population of pepper (Capsicum annuum)

Quantitative trait loci (QTL) for resistance to phytophthora root rot caused by Phytophthora capsici were investigated using two Korean P. capsici isolates and 126 F₈ recombinant inbred lines derived from a cross of Capsicum annuum line YCM334 (resistant parent) and local cv. Tean (susceptible parent). The experimental design was a split plot with two replications. Highly significant effects of pathogen isolate, plant genotype, and genotype × isolate were detected. QTL mapping was performed using a genetic linkage map covering 1486·6 cM of the pepper genome, and consisted of 249 markers including 136 AFLPs (Amplified Fragment Length Polymorphisms), 112 SSRs (Simple Sequence Repeats) and one CAPS (Cleaved Amplified Polymorphic Sequence). Fifteen QTLs were detected on chromosomes 5 (P5), 10 (P10), 11 (P11), Pb and Pc using two data processing methods: percentage of wilted plants (PWP) and relative area under the disease progress curves (RAUDPC). The phenotypic variation explained by each QTL (R²) ranged from 6·0% to 48·2%. Seven QTLs were common to resistance for the two isolates on chromosome 5 (P5); six were isolate‐specific for isolate 09‐051 on chromosomes 10 (P10) and Pc, and two for isolate 07‐127 on chromosomes 11 (P11) and Pb. The QTLs in common with the major effect on the resistance for two isolates explained 20·0–48·2% of phenotypic variation. The isolate‐specific QTLs explained 6·0–17·4% of phenotypic variation. The result confirms a gene‐for‐gene relationship between C. annuum and P. capsici for root rot resistance.     

Evidence of low levels of genetic diversity for the Phytophthora austrocedrae population in Patagonia,Argentina

Phytophthora austrocedrae is a recently discovered pathogen that causes severe mortality of Austrocedrus chilensis in Patagonia. The high level of susceptibility of the host tree, together with the distribution pattern of the pathogen, have led to the hypothesis that P. austrocedrae was introduced into Argentina. The aim of this study was to assess the population structure of P. austrocedrae isolates from Argentina in order to gain an understanding of the origin and spread of the pathogen. Genetic diversity was determined based on amplified fragment length polymorphisms (AFLPs). In total, 48 isolates of P. austrocedrae were obtained from infected A. chilensis trees, representing the geographical range of the host. Four primer combinations were used for the AFLP analysis. Of the 332 scored bands, 12% were polymorphic. Gene diversity (h) ranged from 0·01 to 0·03; the Shannon index (I) ranged from 0·01 to 0·04. A high degree of genetic similarity was observed among the isolates (pairwise S values = 0·958–1; 0·993 ± 0·009, mean ± SD). A frequency histogram showed that most of the isolate pairs were identical. Principal coordinate analysis using three‐dimensional plots did not group any of the isolates based on their geographical origin. The low genetic diversity (within and between sites) and absence of population structure linked to geographic origin, together with the aggressiveness of the pathogen and the disease progression pattern, suggest that P. austrocedrae might have been introduced into Argentina.     

Phenotypic and molecular genetic characterization indicate no major race‐specific interactions between Xanthomonas translucens pv. graminis and Lolium multiflorum

Bacterial wilt of forage grasses, caused by the pathogen Xanthomonas translucens pv. graminis (Xtg), is a major disease of forage grasses such as Italian ryegrass (Lolium multiflorum). The plant genotype‐bacterial isolate interaction was analysed to elucidate the existence of race‐specific responses and to assist the identification of plant disease resistance genes. In a greenhouse experiment, 62 selected plant genotypes were artificially inoculated with six different bacterial isolates. Significant differences in resistance were observed among L. multiflorum genotypes (P < 0·001) and in virulence (intensity of disease symptoms) among Xtg isolates (P < 0·001) using the area under the disease progress curve (AUDPC). No significant genotype‐isolate interaction (P > 0·05) could be observed using linear regression modelling. However, additive main effects and multiplicative interaction effects (ammi ) analysis revealed five genotypes which did not cluster close to the origin of the biplot, indicating specific interactions between these genotypes and some bacterial isolates. Simple sequence repeat (SSR) markers were used to identify marker‐resistance associations using the same plant genotypes and bacterial isolates. The SSR marker NFA027 located on linkage group (LG) 5 was significantly associated with bacterial wilt resistance across all six bacterial isolates and explained up to 37·4% of the total variance of AUDPC values. Neither the inoculation experiment nor the SSR analyses revealed major host genotype‐pathogen isolate interactions, thus suggesting that Xtg resistance, observed so far, is effective across a broad range of different bacterial isolates and plant genotypes.     

Two paths of sequence divergence in the citrus tristeza virus complex

ABSTRACT Comparison of a sampling of complementary DNA (cDNA) sequences from the Florida citrus tristeza virus (CTV) isolates T3 and T30 to the sequence of the genome of the Israeli isolate VT showed a relatively consistent or symmetrical distribution of nucleotide sequence identity in both the 5' and 3' regions of the 19.2-kb genome. In contrast, comparison of these sequences to the sequence of isolate T36 showed a dramatic decrease in sequence identity in the 5' proximal 11 kb of the genome. A cDNA probe derived from this region of the T36 genome hybridized to double-stranded RNA (dsRNA) of only 3 of 10 different Florida CTV isolates. In contrast, analogous probes from T3 and T30 hybridized differentially to the seven isolates not selected by the T36 probe. Primers designed from cDNA sequence for polymerase chain reaction (PCR) selectively amplified these 10 isolates, allowing them to be classified as similar to T3, T30, or T36. In contrast, individual cDNA probes derived from the 3' terminal open reading frames of the T3, T30, and T36 genomes all hybridized to dsRNA from all Florida CTV isolates tested, and PCR primers designed from the T36 capsid protein gene sequence amplified successfully from all isolates. Based on these data, we propose the creation of two groups of CTV, exemplified by the VT and T36 isolates, respectively. Isolates in the VT group, which include isolates VT, T3, and T30, have genomic sequence divergence that is relatively constant in proportion and distribution throughout the genome, and candidate isolates for that group could be considered strains of the same virus. The T36 group is differentiated from the VT group by the highly divergent 5' genomic sequence. This 5' region of the CTV genome, thus, can serve as a measure of the extent of sequence divergence and can be used to define new groups and group members in the CTV complex.     

Molecular analysis suggests that recent <Emphasis Type="Italic">Citrus tristeza virus</Emphasis> outbreaks in Italy were originated by at least two independent introductions

Citrus tristeza virus (CTV) is the causal agent of the most important virus disease of citrus. Numerous CTV isolates differing in biological and molecular characteristics have been reported worldwide. Recently, CTV was detected in Italy in several citrus crops from three separate areas: (1) Cassibile, province of Syracuse; (2) Massafra, province of Taranto; and (3) Belpasso, province of Catania. CTV isolates from Massafra and Cassibile were mild, whereas isolates from Belpasso induced severe symptoms. To study the genetic variation of CTV populations of these areas, 150 samples per area were examined by single strand conformation polymorphism (SSCP) and nucleotide sequence analysis of CTV gene p20. All isolates from the same area showed the same SSCP pattern whereas for each area a different SSCP pattern was obtained. The Massafra and the Cassibile isolates had a nucleotide identity higher than 99% with a mild isolate from Spain and about 92% with the Belpasso isolates, which were similar (identity higher than 99%) to severe isolates from California and Japan. These results suggest at least two independent introductions of CTV in Italy, probably by import of CTV-infected budwoods. Within each area, the virus population was homogeneous suggesting diffusion of CTV by aphid transmission. The GenBank accession numbers of the sequences reported in this paper are: AY262000, AY263360 and AY263361 corresponding to gene p20 of CTV isolates from Massafra, Cassibile and Belpasso (Italy), respectively.     

Pathotypic and genetic diversity in the population of Rhizoctonia solani AG1‐IA causing rice sheath blight in China

Sheath blight, caused by Rhizoctonia solani AG1‐IA, is one of the most serious diseases of rice. In this study, a total of 175 isolates of R. solani AG1‐IA were collected from five rice‐growing regions in China. Pathogenicity tests revealed that all isolates were virulent to five cultivars with different levels of resistance at the rice seedling stage in the greenhouse. There was considerable variation in aggressiveness, and the isolates were classified into three pathotypes based on disease severity, with moderately virulent isolates prevalent in the population. Forty‐three haplotypes were identified based on ITS sequencing, and 39 haplotypes were distinct among isolates. There were high levels of haplotype diversity and nucleotide diversity within the populations of R. solani AG1‐IA. High gene flow (Nm = 1·63–5·22) was detected, consistent with relatively low differentiation between pairs of populations. Five populations were divided into two distinct clusters by the unweighted pair group method with arithmetic mean (UPGMA), and no spatial population differentiation was discernible. The majority (97·8%) of genetic diversity was distributed among isolates within populations, with only 2·2% of the genetic diversity attributed to differences among populations. The star‐like shape of the haplotype network provided evidence of signatures of population expansion in recent history. No significant relationships were found between the genetic diversity and aggressiveness or geographic origin among populations of R. solani AG1‐IA. These results highlight that the population characteristics of R. solani AG1‐IA should be taken into account in evaluating the germplasm resistance of rice cultivars to sheath blight.     

Genetic analysis of the population structure of the rice false smut fungus,Villosiclava virens,in China using microsatellite markers mined from a genome assembly

Studies on population genetics of Villosiclava virens are limited because of the lack of polymorphic markers. Based on a draft genome sequence of isolate HWD‐2 produced in this study, 20 of 403 potential simple sequence repeats (SSR) loci showed polymorphisms in preliminary screening using eight diverse V. virens isolates. Among polymorphic loci, most of them with tetra‐ to hexanucleotide unit motifs showed higher levels of polymorphism than loci with smaller motifs. After testing with 20 polymorphic SSR markers, the 87 isolates of V. virens from eight populations in China showed a high level of genetic diversity, with each as a unique haplotype. This differs from some previous findings showing little to no genetic variation based on random amplified polymorphic DNA and amplified fragment length polymorphism analyses. Among eight populations from major rice production areas of China, the population from Guangxi province in south China showed the highest levels of polymorphism, which led to the speculation that it might be closer to the centre of origin of this pathogen. The northern, central and eastern populations (Jilin, Liaoning, Hubei, Hunan, Jiangxi and Zhejiang), when considered together as a group, showed significant molecular variation compared to the southern populations (Fujian and Guangxi) (Φ_PT = 0·043, P = 0·037). A significant relationship (Mantel test, P = 0·027) but with low correlation (R² = 0·23) was also found between geographic distance and genetic distance. The 20 polymorphic SSR primer pairs designed in this study provide a tool for further research on the population diversity of this emerging fungal pathogen of rice.     

Genotyping Cephalosporium gramineum and development of a marker for molecular diagnosis

This study investigated the genetic variation of 40 isolates of Cephalosporium gramineum, the causal agent of cephalosporium stripe disease of wheat, based on variations in internal transcribed spacers (ITS) and intergenic spacers (IGS) of rDNA. Of the isolates, 29 were from Japan and the rest from the USA and Europe. The ITS region was about 600 bp and almost identical among these isolates. In the IGS region (～5 kbp), restriction fragment length polymorphism analysis detected four genotypes among the 40 isolates. One representative isolate was selected from each of the four genotypes, and the IGS region was sequenced. Attempts to design a genotype‐specific marker based on the size of PCR products amplified with selected primers failed to differentiate among the four genotypes. Alternatively, a species‐specific primer set (CGIGS1 and CGIGS2) was developed that annealed within the conserved region, producing a DNA fragment of about 1·8 kbp. Tests of this primer set on a wide range of other fungi from 11 genera confirmed that it was specific to C. gramineum. This primer set could serve as an effective tool in the molecular diagnosis of C. gramineum and has the potential to assist in a better understanding of the host–pathogen interaction.     

Lack of host specificity of Colletotrichum spp. isolates associated with anthracnose symptoms on mango in Brazil

The aim of the present study was to analyse the genetic and pathogenic variability of Colletotrichum spp. isolates from various organs and cultivars of mango with anthracnose symptoms, collected from different municipalities of São Paulo State, Brazil. Colletotrichum gloeosporioides isolates from symptomless citrus leaves and C. acutatum isolates from citrus flowers with post‐bloom fruit drop symptoms were included as controls. Sequencing of the ITS region allowed the identification of 183 C. gloeosporioides isolates from mango; only one isolate was identified as C. acutatum. amova analysis of ITS sequences showed larger genetic variability among isolates from the same municipality than among those from different populations. fAFLP markers indicated high levels of genetic variability among the C. gloeosporioides isolates from mango and no correlation between genetic variability and isolate source. Only one C. gloeosporioides mango isolate had the same genotype as the C. gloeosporioides isolates from citrus leaves, as determined by ITS sequencing and fAFLP analysis. Pathogenicity tests revealed that C. gloeosporioides and C. acutatum isolates from either mango or citrus can cause anthracnose symptoms on leaves of mango cvs Palmer and Tommy Atkins and blossom blight symptoms in citrus flowers. These outcomes indicate a lack of host specificity of the Colletotrichum species and suggest the possibility of host migration.     

瓜类褪绿黄化病毒山东分离物全基因组序列扩增及分析

为明确分离自山东省寿光市甜瓜上的瓜类褪绿黄化病毒(cucurbit chlorotic yellows virus,CCYV)分离物的全基因组序列信息和遗传变异情况,利用毛形病毒属Crinivirus简并引物进行RTPCR检测,利用RACE技术结合RT-PCR方法克隆CCYV山东分离物2条RNA链的全基因组序列,通过与GenBank中其它地区CCYV分离物的全长序列进行比对分析其同源性,并基于CP基因序列构建系统进化树分析其遗传变异情况。结果表明,山东分离物经RT-PCR检测和测序后确定为CCYV。CCYV山东分离物与其它CCYV分离物的RNA1链和RNA2链的全基因组序列一致性的平均值分别为99.82%和99.88%,且2条链的5′末端均比较保守,没有碱基突变的情况发生;RNA1链3′末端存在2个碱基变异,RNA2链3′末端存在1个碱基变异。CCYV不同地区分离物主要分为3个簇群,其中山东分离物和中国其它地区分离物、日本分离物、苏丹分离物、黎巴嫩分离物和塞浦路斯分离物聚类在一起。研究表明CCYV基因组序列比较保守,该病毒的分化可能与地理来源存在一定的相关性。