Transmission of six ampeloviruses and two vitiviruses to grapevine by Phenacoccus aceris

Grapevine leafroll disease is caused by grapevine leafroll-associated viruses (GLRaVs). These viruses are common in vineyards worldwide and often associated with vitiviruses that are involved in the rugose wood complex of grapevine. Ten mealybug species are known as vectors of one or several of these grapevine viruses, including the apple mealybug Phenacoccus aceris which is widespread in Holarctic regions and able to transmit Grapevine leafroll-associated virus-1 and -3 (GLRaV-1 and -3). Our aim was to characterize the transmission features of leafroll viruses by Phenacoccus aceris in order to better understand the contribution of this mealybug to leafroll epidemics. Results showed that Phenacoccus aceris is able to transmit GLRaV-1, -3, -4, -5, -6, and -9 to grapevine but not GLRaV-7. This is the first report of GLRaV-6 transmission by a mealybug. Also, for the first time it was shown that Phenacoccus aceris could vector vitiviruses Grapevine virus A (GVA) and Grapevine virus B (GVB). First instar nymphs were the most efficient stage in transmitting GLRaV-1, -3, and GVA. This research sheds light on the transmission biology of grapevine viruses by Phenacoccus aceris and represents a step forward to leafroll disease management.     

No evidence of transmission of grapevine leafroll-associated viruses by phylloxera (<Emphasis Type="Italic">Daktulosphaira vitifoliae</Emphasis>)

Grapevine leafroll disease is associated with several species of phloem-limited grapevine leafroll-associated viruses (GLRaV), some of which are transmitted by mealybugs and scale insects. The grape phylloxera, Daktulosphaira vitifoliae (Fitch) Biotype A (Hemiptera: Phylloxeridae), is a common vineyard pest that feeds on the phloem of vine roots. There is concern that these insects may transmit one or more GLRaV species, particularly GLRaV-2, a species in the genus Closterovirus. A field survey was performed in vineyards with a high incidence of grapevine leafroll disease and D. vitifoliae was assessed for acquisition of GLRaV. In greenhouse experiments, the ability of D. vitifoliae to transmit GLRaV from infected root sections or vines to co-planted virus-free recipient vines was tested. There were no GLRaV-positive D. vitifoliae in the field survey, nor did D. vitifoliae transmit GLRaV-1, ?2, ?3, or -4LV in greenhouse transmission experiments. Some insects tested positive for GLRaV after feeding on infected source vines in the greenhouse, however there was no evidence of virus transmission to healthy plants. These findings, in combination with the sedentary behaviour of the soil biotype of D. vitifoliae, make it unlikely that D. vitifoliae is a vector of any GLRaV.     

Viruses and virus diseases of grapevine in Egypt

Surveys for virus and virus-like diseases were carried out in commercial vineyards of the main grapevine-growing areas of Egypt along the river Nile and in recently reclaimed desert lands. The only symptoms observed and identified with reasonable confidence in the field were those of leafroll disease in red-berried cultivars. No virus was transmitted to herbaceous hosts by mechanical inoculation from glasshouse-forced cuttings of about 300 vines (40% of total samples). By contrast, ELISA tests showed that 78% of the assayed European vines (521 out of 664) were infected by one (29%) or more (49%) viruses. Grapevine virus A (GVA) was the most widespread virus (67.9% infection), followed by Grapevine leafroll-associated virus 3 (GLRaV-3) (55.9% infection). All the other viruses tested for were scarcely represented, i.e. Grapevine leafroll-associated virus 1 (GLRaV-1) 1.8% infection, Grapevine leafroll-associated virus 2 (GLRaV-2) 1.4% infection, Grapevine virus B (GVB) (0.6% infection) and Grapevine fleck virus (GFkV) (0.2% infection), or, like Grapevine fanleaf virus (GFLV), were totally absent. The infection rate of native cultivars (86%) was particularly heavy. 'Banaty Abiad' and 'Romy Ahmer', the two major Egyptian cultivars, had infection levels of 78% and 89%, respectively, and 'Fayoumy', the most important cultivar in the Fayoum area, had 96% infection. Totally infected were the tested samples of several minor native cultivars such as 'Farg El-Tair', 'Siwi Abiad', 'Ta'afi', 'Romy Abiad', 'Eswid El-Wady', 'Edkawy' and 'Bez El-Anza'. Slightly better was the sanitary situation of imported European grapevine cultivars (60% infection) and of American rootstocks (11.5% infection). In rootstocks, infection rate by GVA and GLRaV-3 was 5.5%, whereas GVB and GLRaV-1 were only sporadically detected.     

Viruses and virus diseases of grapevine in Lebanon

Grapevines were surveyed for the presence of virus and virus-like diseases in the main viticultural areas of Lebanon (Bekaa valley, Mount Lebanon, South and North Lebanon). Symptoms of rugose wood were observed in vines ofall cultivars and areas surveyed, whereas leafroll was observed only in some vineyards of the Bekaa valley and, to a lesser extent, in South Lebanon on cvs Tfaifihi, Cinsaut and Cardinal. Symptoms of fanleaf and of phytoplasma-induced yellows were also observed with low frequency in the Bekaa valley on wine-grape cultivars. ELISA tests showed that 53% of 1536 Vitis vinifera vines individually checked were infected by one or more viruses. Grapevine trichovirus A (GVA) was the prevailing virus (32.4%), followed by grapevine fleck virus (GFkV) (19.5%) and grapevine leafroll-associated closterovirus 3 (GLRaV-3) (12.4%). Grapevine leafroll-associated closterovirus 1 (GLRaV-l), grapevine trichovirus B (GVB) and grapevine fanleaf nepovirus (GFLV) were also detected to a lesser extent, their incidence ranging between 1.1 and 3.6%.     

Viruses and virus diseases of grapevine in Tunisia

Mature canes were collected from vines in the main grapevine-growing areas in Tunisia (Cape Bon, Bizerte, Ben Arous), from commercial vineyards and mother-plant plots, to assess the presence of virus and virus-like diseases. Biological (mechanical transmission onto herbaceous hosts and grafting onto indicator woody plants) and serological detection (ELISA) methods were applied. ELISA showed that 96.4% of 669 vines tested were infected, most of them (88.1%) by at least two viruses. Grapevine leafroll-associated 3 closterovirus (GLRaV-3) was the most widespread virus (87.9%), followed by grapevine A vitiviras (GVA, 69.4%), grapevine fleck virus (GFkV, 51.9%), grapevine leafroll-associated 1 closterovirus (GLRaV-1, 36.8%), grapevine leafroll-associated 2 closterovirus (GLRaV-2, 19.1%), grapevine fan leaf nepovirus (GFLV, 18.2%) and grapevine B vitiviras (GVB, 14.8%). ELISA tests yielded negative results for grapevine leafroll-associated 7 closterovirus (GLRaV-7) and potato X potexvirus (PVX). The highest infections were found in Bizerte and Cape Bon regions (100 and 99.2%), and in vineyards aged over 20 years (98.5%) as compared with the younger ones (81.1%). Rootstocks in mother-plant plots were practically free from all the viruses tested (1 plant infected out of 81), whereas severe infections were found in Vitis vinifera mother plants (67.4% of 341 samples), in particular table grapes (92.6%) compared with wine grapes (47.9%). In these mother-plant plots, the prevailing viruses were GLRaV-3 (41.3%), followed by GFkV (36.7%), GVA (27.9%), GLRaV-1 (17%) and GLRaV-2 (15.2%). GFLV and GVB were far more limited (1.5 and 0.6%, respectively). The presence of vein necrosis and vein mosaic was ascertained by transmission onto 110R and Vitis riparia indicators, whereas only GFLV was mechanically transmitted onto herbaceous hosts (from about 20% of the samples).     

Detection of Grapevine A vitivirus in Tunisian grapevines

Grapevine A vitivirus (GVA), Grapevine B vitivirus (GVB), Grapevine leaf-roll associated closterovirus 3 (GLRaV3) and Grapevine fanleaf nepovirus (GFLV) were detected by DAS-ELISA in samples of grapevine showing symptoms of rugose wood from different viticultural areas of Tunisia. After optimization of experimental conditions, PCR and IC-RT–PCR were found to be more efficient and sensitive than DAS-ELISA for the detection of GVA.     

Grapevine leafroll-associated viruses and grapevine virus A in selected Vitis vinifera cultivars in northern Italy

The occurrence of grapevine leafroll-associated virus 1 (GLRaV-1), grapevine leafroll-associated virus 3 (GLRaV-3) and grapevine virus A (GVA) was demonstrated in a viticultural region of northern Italy (Emilia-Romagna) using immunoelectron microscopy. Virus incidence was subsequently assessed using ELISA. A total of 60.6% of the 150 clone selections tested, from 18 local Vitis vinifera cultivars, were found to be infected. ELISA did not reveal the presence of grapevine leafroll-associated virus 2 (GLRaV-2) or grapevine leafroll-associated virus 5 (GLRaV-5). GLRaV-1, GLRaV-3 and GVA were found individually and in various combinations. The most common findings were GLRaV-1 alone (25.3%) and associated with GVA (33%). Serological data confirmed that the majority (91%) of the clones known to be affected by grapevine leafroll (GLR), on its own or in association with rugose wood (RW), contained viruses. On the other hand, where the RW phenomenon was present on its own, only 40% of these clones were ELISA-positive. The implications for the biology of GLR and RW are discussed and the complex aetiology of these grapevine diseases is confirmed.     

Viruses of grapevine in Syria

Surveys for virus and virus-like diseases were carried out in commercial vineyards and nurseries in seven different Syrian provinces (Aleppo, Dara'a, As Suwayda, Al Qunaytirah, Homs, Hamah, Tartous). Samples were collected at random from 835 individual vines (735 Vitis vinifera and 100 rootstock accessions) for laboratory testing. Grapevine fanleaf virus (GFLV) , Arabis mosaic virus (ArMV), and Grapevine virus A (GVA) were the only viruses recovered by mechanical transmission to herbaceous hosts. Vein necrosis developed in c. 53% of graft-inoculated 110R indicators and vein mosaic in V. riparia inoculated with material from cv. Corna Alegra. A total of 71% of the ELISA-tested V. vinifera plants (522 out of 735) were infected by one (14.8%) or more (55.8%) viruses. GVA was the most widespread (54.7%), followed by Grapevine leafroll-associated virus 1 (GLRaV-1, 47.3%), Grapevine fleck virus (GFkV, 29.7%), and Grapevine leafroll-associated virus 3 (GLRaV-3, 23.9%). Other economically relevant viruses were scarcer, i.e. Grapevine leafroll-associated virus 2 (GLRaV-2, 9%), GFLV (0.8%) and ArMV (0.1%). The most important Syrian grapevine varieties, i.e. Hellwany, Salty, Balady, and Zeiny, had average infection rates that ranged between 44% and 91%. The highest incidence of infections was observed at Damascus (90%), whereas it ranged between 68% and 79% in the other provinces, except for Hama (36%). Rootstocks were in much better sanitary condition (25% infection). GFkV (22%) was the most common virus, whilst the presence of GLRaV-3 (3%), GLRaV-1, and GFLV (1%) was negligible. Grapevine rupestris stem pitting associated virus (GRSPaV) was detected in 72.3% of the samples by RT-PCR. A high percentage of the GRSPaV-positive vines (80%) induced vein necrosis reactions in 110R, thus confirming the recently established correlation between this virus and vein necrosis.     

Crude garlic extract significantly inhibits replication of grapevine viruses

Efforts to control viral diseases of grapevine include the production of certified material and development of virus-resistant transgenic grapevines. However, effective antiviral agents, once the viruses have infected the plants, are still lacking. This study shows that a crude garlic extract has significant antiviral activity against grapevine viruses. Replication of grapevine leafroll-associated virus 2 (GLRaV-2) was obviously inhibited in grapevine cv. Cabernet Sauvignon calli treated with diluted (1:100) garlic extract. The relative RNA levels of GLRaV-2 and grapevine fleck virus (GFkV) in cv. Summer Black grapevine in in vitro-grown plantlets 10 days after treatment with diluted (1:100) garlic extract were about 22% and 20%, respectively, of that in controls. The viral RNA accumulation of GLRaV-2, GFkV, grapevine virus A (GVA), grapevine fanleaf virus (GFLV) and grapevine rupestris stem pitting-associated virus (GRSPaV) in field-grown grapevine cv. Centennial Seedless plants sprayed with diluted (1:100) garlic extract were about 31–40%, 26–38%, 18–31%, 17–42% and 15–18%, respectively, of that in controls. Moreover, the garlic extract treatment led to a significant decrease in viral RNA accumulation of GLRaV-3, GLRaV-2, GVA, GFkV, GFLV, GRSPaV and grapevine Pinot Gris virus in pot-grown grapevine cv. Shine Muscat plants, and viral disease symptoms in these plants were obviously attenuated. In addition, this extract significantly induced expression of pathogenesis-related protein genes and stimulated activity of antioxidant enzymes in grapevines. Taken together, these results indicate that the crude garlic extract acts as a significant inhibitor against a broad range of grapevine viruses.     

Grapevine virus A in Rheinland-Pfalz: Vorkommen und Bedeutung für den deutschen Weinbau

Grapevine virus A (GVA) is associated with Kober stem grooving disease which belongs to the Rugose wood complex. The virus is frequently found in grapevine affected by leafroll but is not strictly associated with this disease. Probably the virus has a worldwide distribution – but very little information is available about the occurrence of GVA in Rhineland-Palatinate. The detection of GVA was conducted with indexing procedures using Kober 5BB as indicator and serological methods (ELISA). Indexing trials with infected material did not show any symptoms of Kober stem grooving two years after wooden grafting and five years after green grafting. The most suitable tissue to detect GVA serologically was petioles from mature leaves or cortical scrapings from dormant canes. In all experiments the tests with petioles resulted in much higher extinction values compared to the blades. About 46.9% of the 209 samples tested had an infection of GVA, 87.8% in mixed infections together with GLRaV-1 and 5.1% together with GLRaV-3. Only 7.1% of the tested plants were exclusively infected by GVA, none of the other ampelo- and closteroviruses could be detected. None of the GVA positively tested plants showed any symptoms of Rugose wood. The results indicated that the importance of a GVA infection on vines in Germany seems to be extremely low.     

Identification of herbaceous hosts of the <Emphasis Type="Italic">Grapevine Pinot gris virus</Emphasis> (GPGV)

Grapevine Pinot gris Virus (GPGV) is a single stranded RNA of the genus Trichovirus infecting grapevine (Vitis vinifera) and associated with stunting, chlorotic mottling and leaf deformation symptoms. During a monitoring of GPGV infection in vineyards of the Trentino region in Italy, we have detected the virus in the herbaceous plants Silene latifolia subsp. Alba (Mill.) (bladder campion) and Chenopodium album L. (white goosefoot), which showed symptoms of viral infection. The full-length GPGV RNA genome, amplified from these infected hosts, was sequenced and a phylogenetic analysis revealed that its closest relative is the strain SK13, recently isolated in Slovakia. Our results indicate that herbaceous plants can be considered as a reservoir for the GPGV virus. This finding is important for studying the epidemiological aspects of GPGV disease and to formulate appropriate control measures.     

Phytosanitary status of grapevine in Montenegro

During 2006 and 2007, a survey on the incidence and distribution of fourteen grapevine viruses was carried out in the Skadar Lake basin, one of the two main grapevine‐growing areas of Montenegro. In total 165 samples were collected from four red (‘Vranac’, ‘Krato?ija’, ‘Merlot’ and ‘Cardinal’), two white (‘Chardonnay’ and ‘Rkaciteli’) and a few unknown grapevine varieties in the vicinity of Podgorica and Bar. The phytosanitary status of the collected samples was analysed by DAS‐ELISA and the presence of Grapevine fanleaf virus (GFLV), Grapevine leafroll‐associated virus 1 (GLRaV‐1), Grapevine leafroll‐associated virus 2 (GLRaV‐2) and Grapevine leafroll‐associated virus 3 (GLRaV‐3) was confirmed in some of them. The most frequently found virus in assayed samples was GLRaV‐3 (54.5%), followed by GFLV (23%), GLRaV‐1 (20%) and GLRaV‐2 (0.6%). These serological analyses showed the absence of Grapevine leafroll‐associated virus 6 (GLRaV‐6), Grapevine leafroll‐associated virus 7 (GLRaV‐7), Raspberry bushy dwarf virus (RBDV), Strawberry latent ringspot virus (SLRSV), Tomato ringspot virus (ToRSV), Raspberry ringspot virus (RpRSV), Arabis mosaic virus (ArMV), Tobacco ringspot virus (TRSV), Tomato black ring virus (TBRV) and Cherry leaf roll virus (CLRV) from all tested samples.     

High prevalence of viruses in table grape from Spain detected by real-time RT-PCR

Table grapes from one of the most important growing area in Spain (Vinalopó, Alicante) protected by the Designation of Origin “Vinalopó bagged table grape”, were surveyed and analysed to determine the prevalence of the five viruses included in the Spanish certification program: Arabis mosaic virus (ArMV), Grapevine fanleaf virus (GFLV), Grapevine fleck virus (GFkV), Grapevine leafroll associated virus-1 (GLRaV-1) and Grapevine leafroll associated virus-3 (GLRaV-3). Ninety five sampling points were selected and the position of grapevine plants georeferenced. Samples were collected in two different vegetative periods and analyses were performed by ELISA and real-time RT-PCR. Purified RNA and immobilized viral targets from plant extracts on nylon membranes were used in parallel assays as templates for PCR assays. In order to analyse these five viral species by real-time RT-PCR, new specific primers and TaqMan probes were designed for detection of ArMV and GFkV. Real time RT-PCR from purified RNA was more sensitive than spot version and ELISA tests. The most prevalent virus was GFLV (95.8%) followed by GLRaV-3 (94.7%), GLRaV-1 (66.3%) and GFkV (65.3%). ArMV was not detected in any sample. The high level of viral infections and the presence of mixed infections suggest that initial infected plant material and uncontrolled traffic of propagation material have played an important role in the spread of viruses.     

<Emphasis Type="Italic">In situ</Emphasis> localization of <Emphasis Type="Italic">Grapevine fanleaf virus</Emphasis> and phloem-restricted viruses in embryogenic callus of <Emphasis Type="Italic">Vitis vinifera</Emphasis>

In grapevine, somatic embryogenesis is particularly effective in eliminating several important virus diseases. However, the mechanism whereby regenerated somatic embryos are freed of the viruses is not clear. The distribution of Grapevine fanleaf virus (GFLV), Grapevine leafroll-associated virus-3 (GLRaV-3) and Grapevine virus A (GVA) in embryogenic callus of grapevine was investigated by in situ hybridization using digoxygenin-labelled oligonucleotide probes. Four months after culture initiation, in callus originated by GFLV-infected explants we observed a mosaic of infected and uninfected cells, with high concentrations of viruses in some cell groups in peripheral zones of the callus. In addition some abnormal somatic embryos showed a high hybridization signal. In callus originated by GVA- and GLRaV-3-infected explants the viruses were concentrated in few cells surrounded by areas of virus-free cells. The two viruses were generally localized in different clusters of cells inside the callus and the levels of infection were lower than those observed in GFLV-infected callus. No virus was detected in callus nor in somatic embryos after 6 months of culture. The results highlight the difficulties of some viruses at stably invading callus tissues and the differential ability of GFLV to spread in the callus cells compared to the phloem-limited viruses.     

The effects of <Emphasis Type="Italic">Gibberella zeae</Emphasis>, <Emphasis Type="Italic">Barley Yellow Dwarf Virus</Emphasis>, and co-infection on <Emphasis Type="Italic">Rhopalosiphum padi</Emphasis> olfactory preference and performance

Insect-borne viruses promote several changes in plant phenotype, which can modify plant-vector interactions in favor of virus survival and dissemination. Although co-infections commonly occur in the field, little is known about their effects on interactions with the vector. The ecological interactions between Barley Yellow Dwarf Virus (BYDV) and its aphid vector, Rhopalosiphum padi, have been investigated extensively, but the vector’s behavior in more complex scenarios has yet to be examined. We assessed olfactory response and performance of R. padi to wheat singly and doubly infected by the pathogenic fungus Giberella zeae and BYDV. Non-viruliferous aphids preferred odors of BYDV-infected wheat over healthy wheat, as previously reported in the literature, and they were still preferentially attracted to BYDV-infected plant during co-infection. However, around 35% more non-viruliferous aphids chose healthy wheat over G. zeae-infected wheat. Viruliferous aphids did not show any preference to the treatments. BYDV-infected wheat was a superior host than healthy wheat for the aphids whose population increased in 25%. We observed a synergistic effect of the co-infected wheat, which was the best host for aphids, and promoted an elevation of 42% on population growth. Our results indicate that co-infection might be beneficial for virus spread as does not interfere with aphid olfactory preference and provides greater colony growth than in singly infected plants.     

Transmission of grapevine leafroll-associated closteroviruses by Pseudococcus longispinus and P. calceolariae

The transmission of two closteroviruses associated with grapevine leafroll, GLRaV-1 and GLRaV-3, from grapevine to grapevine by the mealybugs, Pseudococcus longispinus and P. calceolariae (Homoptera: Pseudococcidae) was studied. Controlled transmission experiments using the first and third instars of each insect were conducted twice during the 1993–94 growing season to investigate the consequence of virus accumulation within the donor vine leaf tissue on the incidence of virus transmission to healthy recipient vines. Transmission of GLRaV-1 and GLRaV-3 was determined by ELISA testing recipient vines in July 1994 and March 1995. GLRaV-3 was transmitted to recipient vines by P. longispinus and P. calceolariae first instars only. An increase in virus titre within the season did not significantly alter the transmission rate of GLRaV-3 by either P. longispinus or P. calceolariae first instars. P. longispinus and P. calceolariae failed to transmit GLRaV-1 to recipient vines.     

新疆三种葡萄病毒RT-PCR检测及序列分析

为研究新疆葡萄中沙地葡萄茎痘伴随病毒(Grapevine rupestris stem pitting associated virus,GRSPa V)、葡萄斑点病毒(Grapevine fleck virus,GFk V)及葡萄病毒A(Grapevine virus A,GVA)的发生情况和新疆分离株系统进化关系,分别克隆3种病毒新疆分离株部分基因区域,应用RT-PCR对新疆64份葡萄样品中上述3种病毒进行检测,并进行系统进化分析。结果显示,GRSPa V、GFk V和GVA的检出率分别为31.3%、62.5%和25.0%。新疆GRSPa V分离株(KJ801847)与美国GRSPa V分离株(AY368590)同源性达96.59%;新疆GFk V分离株(KJ801846)与日本GFk V分离株(AB222861)及中国辽宁GFk V分离株(JF927942)的同源性分别为91.70%和91.03%;新疆GVA分离株(KJ801845)与波兰GVA分离株(JN860997)同源性为93.88%,与中国四川GVA分离株(HQ671655)及辽宁GVA分离株(FJ445220)的同源性分别为92.92%和89.53%。表明3种葡萄病毒在新疆发生比较普遍,且新疆分离株与国内其它地方的分离株存在较大差异。     

<Emphasis Type="Italic">Grapevine virus A</Emphasis> transmission by larvae of <Emphasis Type="Italic">Parthenolecanium corni</Emphasis>

Grapevine virus A (GVA, Vitivirus) was transmitted experimentally by first and second instars of the scale insect Parthenolecanium corni from grapevine to grapevine and to the herbaceous host Nicotiana benthamiana. This is the first report of GVA transmission by P. corni. Grapevine leafroll-associated virus-1 (Ampelovirus) was always present in the donor grapevines and, in every case, GVA was transmitted simultaneously with this ampelovirus from grapevine to grapevine, suggesting possible interactions between the two viruses for transmission.     

Genetic diversity in the 3' terminal 4.7-kb region of grapevine leafroll-associated virus 3

Grapevine leafroll-associated virus 3 (GLRaV-3; Ampelovirus, Closteroviridae), associated with grapevine leafroll disease, is an important pathogen found across all major grape-growing regions of the world. The genetic diversity of GLRaV-3 in Napa Valley, CA, was studied by sequencing 4.7 kb in the 3' terminal region of 50 isolates obtained from Vitis vinifera 'Merlot'. GLRaV-3 isolates were subdivided into four distinct phylogenetic clades. No evidence of positive selection was observed in the data set, although neutral selection (ratio of nonsynonymous to synonymous substitution rates = 1.1) was observed in one open reading frame (ORF 11, p4). Additionally, the four clades had variable degrees of overall nucleotide diversity. Moreover, no geographical structure among isolates was observed, and isolates belonging to different phylogenetic clades were found in distinct vineyards, with one exception. Considered with the evidence of purifying selection (i.e., against deleterious mutations), these data indicate that the population of GLRaV-3 in Napa Valley is not expanding and its effective population size is not increasing. Furthermore, research on the biological characterization of GLRaV-3 strains might provide valuable insights on the biology of this species that may have epidemiological relevance.