Assessment of Colletotrichum gloeosporioides as a biological control agent for management of hemlock dwarf mistletoe (Arceuthobium tsugense)

The ascomycete Colletotrichum gloeosporioides was tested for biological control of Arceuthobium tsugense on western hemlock (Tsuga heterophylla) in British Columbia (BC), Canada. A field trial was conducted near Nanaimo, BC, using C. gloeosporioides isolate PFC 2415 in three treatments applied in late August 2002. The treatments consisted of C. gloeosporioides formulated with ‘Stabileze’ (incorporation of fungi in a water‐absorbent starch matrix with oil and sucrose, then granulating the matrix with hydrated silica) which were sprayed on (1) intact mistletoe swellings, (2) swellings on which all mistletoe shoots had been cut at 0.5 cm from the base of the shoot and (3) C. gloeosporioides formulated in a sucrose and gelatin preparation sprayed on intact mistletoe swellings. The ultimate goal of this research was to substantially reduce the ability of the treated dwarf mistletoe plants to produce seed inoculum. The ‘Stabileze’ and sucrose–gelatin treatments formulated with C. gloeosporioides reduced the current berry crop by 36.8 and 40.5%, respectively (p < 0.05). While the results for shoots appeared promising, heavy background infection and/or secondary infection, especially on controls, limited the ability to detect clear treatment effects. Careful culturing from various live and dead host tissues showed that C. gloeosporioides was unable to invade and kill the mistletoe endophytic system within the living xylem and phloem of the host.     

Transformation of western hemlock (Tsuga heterophylla) tree crowns by dwarf mistletoe (Arceuthobium tsugense,Viscaceae)

Dwarf mistletoes (Arceuthobium species) are arboreal, hemiparasitic plants of conifers that can change the structure and function of the tree crown. Hemlock dwarf mistletoe (Arceuthobium tsugense subsp. tsugense) principally parasitizes western hemlock (Tsuga heterophylla) and effects 10.8% of all western hemlock trees in Oregon, USA. In this study, we climbed 16 western hemlock trees (age 97–321 years, height 33–54.7 m) across a gradient of infection (0%–100% of branches infected) and measured occurrence of all dwarf mistletoe infections, dwarf mistletoe caused deformities, foliage, branch and crown metrics, and sapwood area. We then modelled over 25 different response variables using linear and generalized linear models with three metrics of severity as explanatory variables: total infection incidence, proportion of all live branches infected, and proportion of all live, infected branches with 33 per cent or more foliage distal to infection. A strong effect of dwarf mistletoe intensification was the reduction of branch foliage and an increase in the proportional amount of foliage distal to infections, with severely infected trees having the majority of foliage distal to infections. Increasing severity led to an apparent crown compaction as crown volumes decreased and became increasingly comprised of deformities. Sapwood area was unrelated to infection severity. Branch length and diameters were unrelated to increasing infection severity despite severely infected branches supporting 1–70 infections. The most severely infected tree had 3,615 individual plants in the crown. Our results suggested that shifts in crown structure and branch deformation, foliage amount, and foliage distal to infection, reflected a likely reduction of capacity for tree growth that coincided with a hypothesized increase in resource demand by dwarf mistletoe plants as infection severity intensified.     

Susceptibility of red fir and white fir to fir dwarf mistletoe (Arceuthobium abietinum) in California

Fir dwarf mistletoe (Arceuthobium abietinum, Viscaceae) is a damaging parasite of true firs (Abies spp.) in California. In the Sierra Nevada Mountains, fir dwarf mistletoe consists of two special forms (formae speciales, f. sp.) which are morphologically similar, but exhibit a high degree of host specificity: A. abietinum f. sp. concoloris whose principal host is Sierra white fir (Abies lowiana) and A. abietinum f. sp. magnificae which only parasitizes red fir (Abies magnifica). Although field observations and cross‐inoculation studies have consistently supported the extreme host specialization demonstrated by these special forms of fir dwarf mistletoe, little quantitative data from mixed red fir/white fir stands infested with fir dwarf mistletoe have been available. Therefore, I sampled 20 mixed conifer stands dominated by red and white fir and infested with f. sp. concoloris (8 stands) or f. sp. magnificae (12 stands) from near Mount Shasta to the southern Sierra Nevada Mountains near Sherman Pass. Infection of red and white fir was evaluated using temporary circular plots (0.012 ha) established around large, severely infected trees and with rectangular transects placed within fir dwarf mistletoe‐infested stands. In each plot and transect, live trees >5 cm in diameter at breast height were assigned a dwarf mistletoe rating (6‐class system). A total of 55 plots and 51 transects were established in the 20 stands, and over 1,900 red firs and 1,700 white firs were sampled. Ninety‐nine per cent of the red firs and 98% of the white firs were infected in stands infested with f. sp. magnificae or f. sp. concoloris, respectively. In contrast, no infection of red fir was observed in stands infested with f. sp. concoloris and no infected white firs were observed in stands infested with f. sp. magnificae. These data provide further support that the special forms of fir dwarf mistletoe are extremely host specific in California.     

Effects of Mexican dwarf mistletoe (Arceuthobium vaginatum subsp. vaginatum) on the growth of Pinus cooperi in Durango,Mexico—A case study

Mexican dwarf mistletoe (Arceuthobium vaginatum subsp. vaginatum, Viscaceae) is the most widespread and damaging parasitic plant in Mexico. It parasitizes 10 species of pines (Pinus spp., Pinaceae) as principal hosts, including Pinus cooperi, one of the economically most important pines in the state of Durango. As a case study, we used stem analysis to estimate the effects of Mexican dwarf mistletoe on volume and height growth of dwarf mistletoe‐infected P. cooperi in western Durango (Ejido El Brillante, Municipality Pueblo Nuevo). This case study sampled a total of 48 trees, 12 in each of four infection classes estimated using the 6‐class dwarf mistletoe rating system (DMR): uninfected (DMR 0), lightly infected (DMR 1–2), moderately infected (DMR 3–4) and severely infected (DMR 5–6). Significant reductions in both volume and height growth were found for moderately and severely infected trees when compared to uninfected trees. On average, reductions in volume growth and height growth were as high as 50% and 17%, respectively. The largest growth reductions were for moderately infected trees, but large growth reductions also occurred for severely infected trees. Because of the reduced growth associated with moderate to severe infection, Mexican dwarf mistletoe‐infested pine forests in Durango, where timber production is a high priority, should be managed using harvesting practices that reduce dwarf mistletoe infection, and thereby, increase forest productivity.     

Evidence of resistance to hemlock dwarf mistletoe (Arceuthobium tsugense) in western hemlock (Tsuga heterophylla) clones

Records from western hemlock (Tsuga heterophylla) trees naturally exposed to infection by hemlock dwarf mistletoe (Arceuthobium tsugense) and controlled inoculations of grafted branch scions from a selection of these trees indicated a repeatability of the levels of resistance to the disease in the clonal material. Evidence pointed to a resistance mechanism operating within, rather than outside, the host branch.     

Infection of Arceuthobium americanum by Colletotrichum gloeosporioides and its potential for inundative biological control

Inundative biological control of Arceuthobium americanum occurring on Pinus contorta var. latifolia with the fungus Colletotrichum gloeosporioides was investigated. Isolates of C. gloeosporioides were collected throughout British Columbia, Canada, and one isolate was selected for assessment based on its growth and sporulation in culture. The fungus was formulated using the ‘Stabileze’ method and inoculated onto A. americanum under field conditions. It became established on some replicates and there was a higher incidence of C. gloeosporioides on treated replicates than controls. In some replicates, the treatment reduced fruit production, leading to a decrease in the reproductive capacity of the dwarf mistletoe plant; however, the efficacy was highly variable and not significant.     

Susceptibility of Coulter pine (Pinus coulteri) to western dwarf mistletoe (Arceuthobium campylopodum) in southern California

The susceptibility of Coulter pine (Pinus coulteri) to western dwarf mistletoe (Arceuthobium campylopodum) was evaluated in southern California. Eight stands infested with western dwarf mistletoe and dominated by Coulter and Jeffrey pines (Pinus jeffreyi) were sampled. Infection of Coulter pine was evaluated using circular plots established around large, severely infected Jeffrey pines and with rectangular transects. A total of 25 plots and 10 transects were sampled. In each plot and transect, live trees > 5 cm in diameter at breast height (1.4 m above the ground) were assigned a dwarf mistletoe rating (6‐class system). Nearly 600 Jeffrey pines and more than 400 Coulter pines were evaluated. Infection of Jeffrey and Coulter pines in the plots and transects averaged 96% and 97%, respectively. These data indicate that Coulter pine should be classified as a principal host of WDM and not as a secondary host as previously reported.     

Factors influencing the dispersion of Arceuthobium oxycedri in Central Spain: evaluation with a new null model for marked point patterns

The dwarf mistletoe, Arceuthobium oxycedri, is found on populations of Juniperus oxycedrus, in central Spain. This species can have negative effects on the physiology of its host, including mortality. Understanding the mechanisms that control its distribution and dispersal is critical to assessing its potential for spread. We assessed dwarf mistletoe distribution within a population of J. oxycedrus, including infected and uninfected host individuals. A new null model of parasitic dispersion was built using two dispersal kernel forms that were simulated with lower and upper envelopes for second‐order functions to summarize a point pattern, such as Ripley's K, nearest‐neighbour distribution and pair correlation functions. Nine dispersal scenarios were constructed with half‐bandwidth kernels (10, 20, 30 m) and initial population of infected trees (P₀ = 05, 10 and 20). These scenarios were compared with the observed pattern and evaluated using the goodness‐of‐fit test. Significant differences at short distance (r < 10 m) were found between the observed pattern and simulated patterns, corresponding to the range of seed dispersal of the dwarf mistletoe. Interactions between infected and uninfected hosts patterns at all scales were identified, suggesting that A. oxycedri uses other mechanisms in addition to ballistic seed shooting as secondary dispersal agents to spread to distances greater than 20 m. Given that the seed characteristics facilitate dispersal by adhesion, we infer that spread between host individuals is amplified by seed transport by birds or small mammals.     

Quantifying the effect of pine mistletoe on the growth of Scots pine

Mistletoe infection results in substantial growth losses in mistletoe‐infected forests. This study reports and evaluates the results of retrospective analyses of radial growth of Scots pine (Pinus sylvestris) in relation to the level of infection of pine mistletoe (Viscum album ssp. austriacum). A total of 43 Scots pine trees were destructively sampled from different sites. Of these trees, 14 were uninfected and 29 were infected. Infection classes were determined using six‐class dwarf mistletoe rating system (DMRS). All needle and mistletoe biomass were removed completely and weighed for each sampled tree. Subsamples from needles and all mistletoe biomass were taken to the laboratory for oven‐dried weight determinations. Five‐cm‐thick wood discs were cut from the stem at the breast height (1.3 m) to determine annual basal area increment for the last 25 years. In addition to DMRS, new infection classes were created using mistletoe‐to‐needle biomass (MB/NB) ratio. The results showed that the radial growth losses could be as much as 41% to 64% at different infection levels. The rate of growth loss in relation to DMRS and MB/NB ratio was similar, but with a larger variability in DMRS values. The results showed that both DMRS rating and MB/NB ratio seem to be important for quantifying growth loss on Scots pine trees infected with mistletoe. The results of this study can also be invaluable in modelling the effects of mistletoe on the growth of Scots pine trees.     

Mortality and growth of dwarf mistletoe‐infected red and white fir and the efficacy of thinning for reducing associated losses

In managed forests dominated by true fir (Abies) species, stands are often restocked using understory trees retained during timber harvest, making the effects of dwarf mistletoe (Arceuthobium spp.) infestation on small true fir a concern. This study examined the response of small red (A. magnifica) and white (A. concolor) fir and their dwarf mistletoes (A. abietinum f.sp. magnificae and A. abietinum f.sp. concoloris, respectively) to precommercial thinning treatments in fir‐dominated stands in the Sierra Nevada Mountains of California. Tree diameters and dwarf mistletoe ratings were monitored from 1981 to 2001, and mortality was observed from 1981 to 2006. Red and white fir survival and radial growth decreased significantly with greater mistletoe ratings and increased with larger diameters and live crown ratios. Thinning significantly increased survival and growth of white, but not red fir. Over the course of the study, mistletoe ratings for both fir species did not change significantly in unthinned stands, but increased in thinned stands. However, while statistically significant, increases in mistletoe ratings in thinned stands were gradual and mistletoe ratings did not statistically differ between treatments 20 years post‐thinning. Additionally, thinning did not significantly influence mistletoe spread to uninfected trees, indicating that precommercial thinning in lightly infested red and white fir stands will not likely result in substantial increases in dwarf mistletoe abundance over typical harvesting intervals. Overall, while severe dwarf mistletoe infection ratings reduced tree survival and growth, because ratings remained low, actual losses resulting from mistletoes during this study were minor and will not likely result in substantial economic losses over normal harvesting intervals. This finding indicates that precommercial thinning treatments designed specifically to compensate for mistletoe‐associated losses may not be necessary when managing red and white fir for timber production.     

The effects of Arceuthobium sichuanense infection on needles and current‐year shoots of mature and young Qinghai spruce (Picea crassifolia) trees

Arceuthobium sichuanense is a hemiparasitic angiosperm that infects Qinghai spruce (Picea crassifolia Kom.) in Qinghai province, China, and causes severe damage to spruce forests in Qinghai‐Tibet Plateau. In this study, the impact of A. sichuanense infection on mature and young trees of Qinghai spruce was evaluated by examining needle and current‐year shoot morphology, needle water and nitrogen‐use efficiency (NUE) and needle nitrogen concentration. The most apparent effect of A. sichuanense infection was a significant reduction in both needle size distal to infection and current‐year shoot length in the infected branches (p < 0.001). Per cent reductions in needle and current‐year shoot length were similar between mature and young trees (58.9 vs. 56.3%; 59.7 vs. 62.9%). There was a high degree of correlation in foliar δ¹⁵N values between the dwarf mistletoe and its host trees (R² = 0.9017, p < 0.001), while the foliar δ¹³C values of A. sichuanense were similar to those of infected mature and young spruce trees. The dwarf mistletoe infection also resulted in a significant decrease in host needle N concentration and δ¹³C values (p < 0.001). The per cent reduction in needle N concentration in young trees was nearly twice as much as that in mature trees (20.49 vs. 11.54%), while the per cent reduction in needle δ¹³C values was similar between young and mature trees (?0.98 vs.?1.1‰). The NUE in mature trees was not affected by A. sichuanense infection, but the NUE in young trees was increased by the infection.     

The influence of prescribed crown fire on lodgepole pine dwarf mistletoe (Arceuthobium americanum) populations 33 years post‐fire

Dwarf mistletoes (Arceuthobium spp.) are a group of obligate, hemiparasitic plants that infect numerous species in the Pinaceae in North America. Wildland fire is considered to be the primary natural agent influencing the population and distribution of dwarf mistletoes across landscapes. Based on this understanding, prescribed fire has been suggested as a potential method for dwarf mistletoe sanitation and control; however, experimental work has primarily focused on prescribed surface fire. In this study, we report long‐term impacts of three experimental crown fires on dwarf mistletoe severity in infested lodgepole pine stands in Colorado 33 years post‐fire. The three fires achieved tree mortality rates ranging from 20% to 100%. Our results suggested a significant negative relationship between the amount of fire‐caused tree mortality and future dwarf mistletoe severity. These findings supported the presumed natural role of fire in altering dwarf mistletoe populations, which perhaps exhibits a linear relationship between fire‐caused host tree mortality and future dwarf mistletoe severity.     

Dieback and decline of wild almond (Amygdalus scoparia Spach) in the Harat protected forest of Yazd Province,Iran

Wild almond (Amygdalus scoparia) is a dominant shrub species in mountain forests of the Irano‐Turanian region. Dieback and decline symptoms of wild almond shrubs were first observed in Harat protected forest (Yazd, Iran) in the autumn of 2014. Since then, the incidence and severity of the disease have increased. To study the aetiology and estimate losses from the disease, field studies were conducted in 2016 and 2017. For this purpose, eight stands were selected, in which 24%–99% of the trees were wild almond in their species composition. In total, 50 cankers and 50 infected twigs of wild almond shrubs were collected. Fungal isolates were identified based on morphological and cultural characteristics, as well as sequence data of ITS1 + 5.8S + ITS2 rDNA. Pathogenicity of fungi was checked on detached shoots and evaluated after 40 days by measuring the discoloured lesion length at the inoculation site. Among 681 wild almond shrubs checked, 84.44% showed decline symptoms with different severities; only 15.56% did not show observable disease symptoms. The most frequent colonizers of infected tissues taken from cankers were Wilsonomyces carpophilus (56%), Thyrostroma cornicola (40%) and Collophorina paarla (30%). Infected twigs were extensively colonized by W. carpophilus (36%), Th. cornicola (24%) and Ulocladium consortiale (24%). Other species, such as Endoconidioma populi, Microsphaeropsis olivacea, Trichoderma asperellum, Paecilomyces formosus, Saccothecium rubi, Preussia sp. and Chaetomium globosum, had lower isolation frequencies. W. carpophilus, Th. cornicola, C. paarla and U. consortiale were found as pathogens on detached wild almond shoots. Based on the frequency of the isolates and the pathogenicity tests, four fungi, such as W. carpophilus, Th. cornicola, C. paarla and U. consortiale, are considered serious contributing agents playing a significant role in the dieback and decline of wild almond. All isolated species are reported for the first time on the wild almond shrubs of the world.     

A new leaf disease on Populus euphratica in south‐western Iran

During the winter of 2012, a leaf spot disease was observed on Euphrates poplar (Populus euphratica) in the forest areas of Khuzestan province, south‐western Iran, causing significant damage in the Karun's riverside forests. Symptoms consisted of necrotic dark brown, circular to oval, 5‐ to 10‐mm spots on both surfaces of the leaves. A fungus having distinct dictyospores similar to those produced by Alternaria spp. was observed. The morphological characteristics, as well as the phylogenetic analysis of the internal transcribed spacer (ITS1‐5,8S‐ITS2) region, confirmed the identity of the strains belonging to the species Alternaria alternata. Pathogenicity tests were conducted on the alive leaves of P. euphratica on the young branches, as well as on the detached leaves in Petri dishes, through inoculation with spore suspension. Target spot symptoms similar to those observed in naturally infected leaves were developed on the inoculated leaves seven to 10 days after inoculation in both the inoculation procedures. A. alternata was consistently re‐isolated from the spots. Interestingly, similar symptoms were observed 7 days after detached leaf treatment with droplets of 15‐day‐old fungal culture filtrate, suggesting the production of pathotoxic compounds by the fungus. To our knowledge, this is the first report of A. alternata causing leaf spot on Euphrates poplar in Iran.     

Hymenoscyphus fraxineus can directly infect intact current‐year shoots of Fraxinus excelsior and artificially exposed leaf scars

Hymenoscyphus fraxineus, the causal agent of ash dieback, was inoculated onto intact, unwounded current‐year shoots and leaf scars of 4‐year‐old, potted Fraxinus excelsior seedlings. Pieces of ash wood colonized by the fungus were used as inoculum. Three of 25 (12%) of the inoculated intact shoots and nine of 25 (36%) of the inoculated leaf scars were infected by H. fraxineus and developed typical symptoms of ash dieback, including necrotic lesions on the shoot surface and wood discoloration as well as shoot and leaf wilting distal to the inoculation site. No symptoms occurred on control seedlings, which had been inoculated in the same way but with sterile wood pieces. Visible necrotic lesions on shoots and wood discoloration were statistically significantly longer in proximal than in distal direction from the inoculation site, a pattern which resembles symptoms after natural infection. The ash dieback pathogen was re‐isolated from nine of 12 (75%) of the symptomatic seedlings. These results provide indirect supportive evidence that the fungus infects shoots via leaves and shows that it is able, under experimental conditions using a massive mycelial inoculum, to directly infect intact, unwounded current‐year shoots of its main host in Europe.     

Variation in virulence of Cryphonectria hypovirus 1 in Macedonia

We investigated variation in virulence of Cryphonectria hypovirus 1 (CHV‐1) to the chestnut blight fungus, Cryphonectria parasitica, in Macedonia by inoculating chestnut stems in the field. We inoculated trees with two isolates of C. parasitica, each infected with one of five isolates of CHV‐1, four of which were the same for both fungal isolates. Two virus isolates, [Sk28] and [Sk47], were significantly more virulent than the others when compared in the same fungal host isolates, as measured by reduced canker growth and increased callus formation. Mycelial growth rate in vitro was weakly correlated to canker growth or callus formation and is therefore not a reliable predictor for virulence. We found significant fungus × virus interactions for canker growth and callus formation, which seems due mainly to one virus isolate. Significant interactions were not expected because the two fungal host isolates are members of the same clone that is dominant in Macedonia and most of southeastern Europe. Phenotypic variation for response to viruses, therefore, is greater than variation revealed by the genetic markers used to define clones. More than half of the trees inoculated with virus‐free controls were dead within 2 years, and the 30% still alive after 5 years had cankers with extensive callus formation, indicating that natural virus transmission had occurred after inoculation. In contrast, only 2% of the trees inoculated with virus‐infected isolates were dead after 5 years. Hypoviruses naturally occurring in Macedonia reduce canker development and tree mortality similarly to those in other parts of southern Europe, and therefore, may have good potential for biological control of chestnut blight.     

Infection patterns and hosts of Arceuthobium oxycedri (DC.) M. Bieb. in Slovenia

Eleven mixed populations of Juniperus oxycedrus L. and Juniperus communis L. were inventoried for the presence of parasitic woody species Arceuthobium oxycedri (DC.) M. Bieb. infections. Both J. oxycedrus and A. oxycedri are rare and distributed in marginal populations in Slovenia. To our knowledge, this is the first detailed research on the host species as well as on the infection and spreading patterns of A. oxycedri in Europe. Assessment of the infection rate was based on the Hawksworth six‐class dwarf mistletoe rating system (DMR). The dimensions of A. oxycedri specimens were surprisingly large and often exceeded 25 cm in diameter, the largest even measuring up to 40 cm. Six juniper populations of 11 were infected, and the proportion of infected host individuals in these ranged between 29.17 and 82.93%. The proportion of infected J. oxycedrus specimens in infected populations was 76.56%, while this percentage for J. communis was 54.90%, which is surprisingly high for this species. A different pattern of infection was observed for the two hosts. A typical infection on J. communis was more localized. Usually, only a single infection was present and was most common on the trunk in the middle third of the crown and much less common on the branches. In J. oxycedrus, infections were also most common in the middle third, but also frequent in other areas of the trunk as well as on the branches. Mostly, we observed a dotted pattern of infection with uninfected areas in between. We assume that A. oxycedri will continue to slowly spread in Slovenia, primarily in areas where it is already present. However, its control is probably not yet necessary.     

Growth characteristics and reproductive output of dwarf mistletoe-infected Juniperus polycarpos in Iran

Dwarf mistletoes are parasitic flowering plants that infect conifers, resulting in substantial loss of growth and mortality. Recently, forest managers in Iran are contemplating whether infection of Juniperus polycarpos C. Koch forests by dwarf mistletoe, Arceuthobium oxycedri （DC.） M. Bieb, influences tree vigor and contributes to insuffieient natural regeneration. The present study aimed at assessing the severity of infection and its impact on growth and reproductive output of./., polycar- pos. Infected and uninfected trees （n =20 each） were selected for assess- ment of diameter, height, crown area, and crown volume as well as quantity and quality of cones and seeds. The severity of infection of trees was determined by Hawksworth＇s 6-class dwarf mistletoe rating （DMR） system. The DMR system revealed that 40% of the infected sample trees were lightly infected （DMR =1-2） and 60% were moderately infected （DMR =3--4）. Growth characteristics did not differ significantly （p 〉 0.05） between infected and uninfected trees. However, moderate infec- tion affected the reproductive output of./. polycarpos by significantly （p 〈0.05） reducing the mean number of cones per unit area of the crown, increasing the number of damaged seeds, and reducing seed size and seedgermination capacity. We conclude that reproductive output of J.. poly- carpos is more sensitive than growth characters to moderate infection by juniper dwarf mistletoe, and this might partly account for poor natural regeneration.     

Relationships between the number of dwarf shoots of<Emphasis Type="Italic"> Larix decidua</Emphasis> Mill. damaged by different insect species in the Ojców National Park in southern Poland

A study was conducted on the interrelationships between the number of dwarf shoots of Larix decidua Mill. damaged by insects: Adelges laricis Vall. Coleophora laricella Hbn., Spilonota laricana Hein. and Dasineura kellneri (Hensch.). The study made use of dwarf shoots collected at three sites in the Ojców National Park, southern Poland during 2002–2003. A total of 240 branches with 19,239 dwarf shoots, collected from 60 larches were designated to the study. The data obtained were subjected to statistical analysis using the Spearman rank correlation coefficient and the Students t-test at the 0.05 level. No unequivocal correlation was found between the relationships.