Characterization and pathogenicity of six Cytospora strains causing stem canker of wild apple in the Tianshan Forest,China

Cytospora species are capable of causing destructive cankers of stems belonging to a wide range of woody plant species. In severe cases, cankers may lead to dieback of twigs and branches. Little is known about the Cytospora species causing canker disease of wild apple (Malus sieversii) trees in the Wild Fruit Forest Reserve in Tianshan Forest, Xinjiang Uygur Autonomous Region, China. In this study, six Cytospora isolates belonging to two species were isolated from cankerous lesions of wild apple twigs. Based on multi‐locus phylogenetic analysis using three DNA markers (ITS, tef1‐α and tub2) and morphological characterization, these isolates were identified as Cytospora mali and Cytospora parasitica. Temperature trials (15, 20, 25 and 30°C) showed that the optimal growth temperature for six isolates was 25°C. At a variety of temperatures, C. mali isolates tended to grow faster than isolates of C. parasitica, with the C. mali isolate, EGI1 performing better than others with regard to growth rate. Morphological observations showed that these species exhibited a single locule without conceptacles, and the conidia length was 3–5 μm. In vitro inoculation trials of twigs and leaves of M. sieversii seedlings revealed that the C. mali isolates were highly virulent phytopathogenic fungi, whereas the C. parasitica isolates were less virulent. The isolate EGI1 was the most virulent isolate among the six isolates. This paper presents the first report of pathogenic Cytospora spp. of the M. sieversii Tianshan Wild Fruit Forest Reserve of Yili, Xinjiang in China. It will aid in the understanding of how apple tree cankers are induced and provide disease management guidelines for M. sieversii forest conservation.     

Fusarium species in declining wild apple forests on the northern slope of the Tian Shan Mountains in north‐western China

Wild apple forests in the Tian Shan Mountains in north‐western China have been adversely affected by an unknown disease in recent years. Symptoms attributed to this disease that affects wild apple trees include xylem browning and dieback which are suggestive of infection by Fusarium species. Therefore, the research team conducted the first survey for Fusarium in the afflicted wild apple forests. Twig samples with symptoms of xylem browning and dieback were collected in the Xinyuan, Gongliu, Yining and Huocheng Counties of Xinjiang Uighur Autonomous Region in China. Based on phylogenetic analyses and morphological observation, sixty strains of Fusarium accounted for 48% of the total number of fungi isolated from samples were subsequently classified into six species including twenty‐four F. avenaceum, seventeen F. solani, ten F. tricinctum, five F. proliferatum, two F. sporotrichioides and two unfamiliar Fusarium sp. 1. The five previously known species of Fusarium were then tested for pathogenicity to leaves and twigs in vitro. The results indicated that all of the species, except for F. tricinctum, can cause obvious lesions on the leaves of host plants and on the twigs of Fuji and wild apple. This is the first report of Fusarium species pathogenicity in Xinjiang wild apple forests, confirming a new host for these pathogens in this study.     

Diplodia corticola and Phytophthora cinnamomi: the main pathogens involved in holm oak decline on Caprera Island (Italy)

Since 2008, severe and widespread tree decline and mortality has been observed at the main growing Quercus ilex L. (holm oak) forest on Caprera Island, Italy. To clarify the symptomatology and aetiology of this phenomenon, field surveys and isolations from symptomatic trees were carried out in summer 2010. Affected trees exhibited crown thinning, branch dieback, sunken cankers, epicormic shoots, exudates on branches and trunk, root losses and sudden death symptoms. Four fungal species belonging to Botryosphaeriaceae family, namely Botryosphaeria dothidea, Diplodia corticola, D. seriata and Neofusicoccum parvum, were isolated from cankers on trunk and branches, whereas three species of Phytophthora, namely P. cinnamomi, P. cryptogea and P. gonapodyides, were isolated from fine roots and rhizosphere soil samples. Isolates were identified using both morphological analysis and DNA‐based techniques. Pathogenicity trials on holm oak seedlings showed that all the isolated species are pathogenic. D. corticola proved to be the most aggressive species. Our results provide the first evidence for a combined involvement of D. corticola and P. cinnamomi in the aetiology of holm oak decline in Italy and suggest that these pathogens are not only important contributing factors in the onset of long‐term tree decline, but also may cause the rapid devastation of extensive oak ecosystems.     

Phytophthora pseudosyringae associated with the mortality of Nothofagus obliqua in a pure stand in central‐southern Chile

Mortality of Nothofagus trees in the southern‐central Chile region has been observed for over 30 years. A field survey conducted in 2013 detected partial defoliation and bleeding cankers on Nothofagus obliqua in a pure stand in the Nahuelbuta coastal ranges of the Biobío region. A Phytophthora sp. was isolated from stem cankers and soil samples around symptomatic N. obliqua trees: All isolates were identified as Phytophthora pseudosyringae. These isolates were pathogenic on 1‐year‐old N. obliqua and Nothofagus alpina, and on detached twigs of adult N. obliqua and Nothofagus dombeyi trees. This paper is the first to report association and pathogenicity of P. pseudosyringae with N. obliqua, N. alpina and N. dombeyi native to the Biobío region of Chile. The potential of P. pseudosyringae to cause damage in natural Nothofagus stands in Chile must be determined.     

Canker of cypresses caused by Coryneum cardinale Wag. in Britain

Coryneum canker occurs widely on Cupressus macrocarpa in England. Mycelial inoculations caused perennial cankers on Cupressocyparis leylandii and Chamaecyparis nootkatensis, severe but annual cankers on Thuja plicata and death of twigs on Juniperus communis. Chamaecy-paris lawsoniana rapidly overcame initial infection. Conidial inoculations on C. leylandii caused cankers. The potential of the disease in Britain and its control are discussed and aids to the identification of the fungus in culture given.     

Brenneria spp. and Rahnella victoriana associated with acute oak decline symptoms on oak and hornbeam in Iran

Decline diseases of forest trees are complex syndromes not attributable to single causal factors. In Iran, symptoms of decline disease have been observed in a number of native forest species including Quercus castaneifolia (chestnut‐leaved oak), Q. brantii (Persian oak) and Carpinus betulus (hornbeam). The symptoms are prevalent in the northern forests and the Zagros mountain forests. There are parallels between the disease in Iran and acute oak decline (AOD) reported in the UK, specifically the presence of weeping cankers, which have been attributed to a polybacterial complex wherein Brenneria goodwinii is considered a key necrogen. Based on the AOD symptomatology, and as a first step towards discovering potential causal agents of the stem weeping symptoms of affected trees in Iran, necrotic tissues were tested primarily for the presence of B. goodwinii. Symptomatic Q. castaneifolia and C. betulus from the Mazandaran Province and symptomatic Q. brantii from Kohgiluyeh and Boyerahmad Province were sampled. Isolation and culture on a selective medium yielded uniform bacterial colonies. Isolates were characterized using phenotypic and genotypic (DNA sequencing) tests. The isolates were phenotypically identical to members of Pectobacteriaceae and Yersiniaceae, specifically Brenneria and Rahnella spp. The nucleotide sequences of the 16S rRNA and housekeeping genes gyrB, infB and atpD (MLSA) amplified via PCR demonstrated that the isolates from the trees in Iran were indeed B. goodwinii, B. roseae subsp. roseae, Rahnella victoriana and an unknown species of Brenneria. Most bacteria isolated from non‐symptomatic trees were Gram‐positive, and Pseudomonas spp. were dominant, but bacterial species isolated from the diseased trees were not detected in healthy trees. Hypersensitivity response tests were positive, but inoculation on saplings was more variable with internal necrosis developing only once in the test period. Therefore, further testing is required. This is the first report of the incidence of B. goodwinii, B. roseae subsp. roseae, R. victoriana and Brenneria sp. associated with acute oak decline‐like symptoms on Q. castaneifolia, Q. brantii and C. betulus across the western forests of Iran and in the world.     

Eutypella canker of maple: first report from Germany and situation in Austria

Eutypella canker of maple, caused by Eutypella parasitica (which is native to North America), is reported for the first time from Germany. From 2013 to 2015, this perennial canker disease was recorded on 105 maple trees in Munich. Six maple species were affected: Acer pseudoplatanus, A. campestre, A. platanoides, A. cappadocicum, A. heldreichii ssp. trautvetteri and A. hyrcanum. Occurrence on the latter three species represents new host records for E. parasitica. In Austria, Eutypella canker was newly discovered on two trees at a second locality in 2011, and it is now known to occur on seven A. pseudoplatanus trees at two localities, which are separated nearly 150 km. A. pseudoplatanus was the most frequent host of E. parasitica in Munich and Austria, which is in agreement with previous studies in Europe. The identity of the causative pathogen as E. parasitica was verified by ITS rDNA sequencing of fungal cultures obtained from cankers in Munich and at both Austrian localities. The presence of large and old cankers in both countries suggests that introduction of E. parasitica dates back a long time, probably several decades. The new records of Eutypella canker in Germany and Austria show that the disease is more widely distributed in central Europe than previously recognized.     

A twig blight of understorey European beech (Fagus sylvatica) caused by soilborne Phytophthora spp.

During and after prolonged periods of rainfall in late spring, blighted young twigs of European beech (Fagus sylvatica) were frequently observed in several beech stands in south‐western and southern Germany. Long and short shoots of young understorey trees or lower branches up to 1.5 m above the soil level were affected. Symptoms also occurred regularly on twigs in heights up to 2 m and more above the ground. Necroses usually expanded within the current year’s tissue and often also reached into the previous year’s wood. Ponding rain water in the stands or along forest roads or open soil seemed to promote the disease. Of a total of 54 symptomatic twigs collected in four stands, 37 revealed Phytophthora isolates, of which 33 were P. plurivora and four were P. cambivora. Both species caused extensive lesions on beech twigs in laboratory pathogenicity tests. Patterns of the disease indicated that these pathogens, generally considered soilborne species, in most cases are transmitted from the soil to above‐ground parts of the trees via rain splash. In larger heights, however, other vectors such as snails might be responsible for transmission. Although Phytophthora spp. are well known as causal agents of seedling blight as well as root and cambium rot and aerial bleeding cankers of mature beech, to our knowledge this is the first report of a twig blight in beech associated with soilborne Phytophthora spp. In particular in periods of high precipitation, this disease might pose an additional threat to Central European beech forests, especially endangering the success of artificial and natural regeneration of beech in affected stands.     

Does Pythium anandrum contribute to the dieback of sessile oak (Quercus petraea) in Turkey?

During investigations into the causes of oak decline in six Regional Forestry Directories of the Black Sea Region of Turkey, Phytophthora spp. were obtained from one region and Pythium spp. were collected from three regions. The most frequently isolated Pythium species, Pythium anandrum, when inoculated on stem bases, caused longer cankers than two isolates of both Phytophthora plurivora and 2 of the P. quercina. This is the first report of Pythium anandrum causing disease on sessile oak. P. anandrum may contribute oak decline in Turkey.     

Diversity of Cryphonectria parasitica in callused chestnut blight cankers on European and American chestnut

Infection of American and European chestnuts with the chestnut blight fungus Cryphonectria parasitica results in the formation of cankers, lesions caused by the growth of mycelia within bark tissue of the host plant. Infection of the fungus with Cryphonectria hypovirus 1 (CHV‐1) results in conversion of the mycelial phenotype from virulent to hypovirulent, thus allowing production of callus around cankers as a reaction by infected trees, rendering active into inactive cankers. In this study, we sampled one USA and six European chestnut stands and assessed frequency of hypovirulent C. parasitica and diversity of vegetative compatibility (vc) types present in calluses and randomly sampled cankers. Callused cankers on C. dentata at West Salem in the USA yielded significantly more hypovirulent C. parasitica isolates compared with four sampled populations on C. sativa, while all six sampled European populations did not show any statistically significant differences among themselves. We observed no correlation between hypovirulence frequencies in randomly sampled cankers and calluses, as well as no correlation of C. parasitica vc type diversity in calluses and residential populations of the fungus. Furthermore, even though we have observed calluses with more than one vc type, they do not occur regularly. Even when present in C. parasitica populations with high vc type diversity, no more than three different vc types were observed in a single callus.     

Previously unrecorded low‐temperature Phytophthora species associated with Quercus decline in a Mediterranean forest in eastern Spain

Oak decline has been a serious problem in Europe since the beginning of the twentieth century. In south‐west Spain, Quercus ilex and Q. suber are the main affected species, and their decline has been associated with Phytophthora cinnamomi. During the last 10 years, a severe decline of Q. ilex and Q. faginea accompanied by a significant decrease in the production of acorns affecting natural regeneration was observed in the eastern part of the Iberian Peninsula. Therefore, the aim of this study was to investigate the possible involvement of Phytophthora spp. in the decline. A forest in the Natural Park ‘Carrascar de la Font Roja’ in Comunidad Valenciana (eastern Spain), which is dominated by Q. ilex and Q. faginea, was surveyed during 2010–2011. Symptomatic trees showed thinning and dieback of the crown, withering of leaves and death. An extensive loss of both lateral small woody roots and fine roots and callusing or open cankers on suberized roots were observed. Soil samples containing fine roots were baited using both Q. robur leaves and apple fruits. Six Phytophthora species were isolated: P. cryptogea, P. gonapodyides, P. megasperma, P. quercina, P. psychrophila and P. syringae. These are the first records of P. quercina and P. psychrophila on Q. faginea, of P. quercina in Spain and of P. psychrophila in mainland Spain. A soil infestation trial was conducted for 6 months under controlled conditions with 1‐year‐old seedlings of Q. ilex and Q. faginea. Phytophthora cinnamomi was included in the pathogenicity test for comparison. The results showed that Q. ilex seedlings were generally more susceptible to infection than Q. faginea with P. cinnamomi being the most aggressive pathogen to both oak species. The two most commonly isolated Phytophthora species, P. quercina and P. psychrophila, also proved their pathogenicity towards both Q. ilex and Q. faginea.     

Chrysoporthe puriensis causing canker and mortality in Pleroma mutabile in the Atlantic Forest,Brazil

Fungi of the Cryphonectriaceae family are globally known to be tree pathogens. In Brazil, several species of Chrysoporthe have been found causing stem and branch cankers in Pleroma (= Tibouchina) spp. Recently, Chrysoporthe puriensis was described as a new species causing stem and branch cankers in Pleroma granulosum, Pleroma candolleanum, and Pleroma heteromallum, all native species of the Melastomataceae family. During an investigation to collect isolates of the Cryphonectriaceae family in an important Brazilian biome, the Atlantic Forest, in Serra do Mar, structures typical of Chrysoporthe species were found in a different host, Pleroma mutabile. Fruiting bodies present in the bark of these trees were collected and isolated. The isolates were submitted on morphological characterization and phylogenetic analyses of the internal transcribed spacer (ITS) and β-tubulin gene regions using Maximum likelihood, Bayesian inference and Maximum parsimony methods. The isolates collected, for this study, were identified as C. puriensis. Pathogenicity tests on seedlings of Pleroma species and Eucalyptus clones revealed C. puriensis can infect and cause canker in these plant species as mortality. The results demonstrate the importance of delimiting the C. puriensis collection range to track its dissemination in other hosts. No host specificity was observed in the inoculation tests, suggesting this is an important finding, the pathogen causes diseases and mortality in several plants of the Atlantic Forest. Additionally, the pathogen can affect others hosts, such as Eucalyptus clones in commercial plantations.     

Pathogenicity to alder of Phytophthora species from riparian ecosystems in western Oregon

Described as one of the most destructive pathogens of agricultural crops and forest trees, Phytophthora is a genus of microorganisms containing over 100 known species. Phytophthora alni has caused collar and root disease in alders throughout Europe, and a subspecies has recently been isolated in North America. Reports of canopy dieback in red alder, Alnus rubra, prompted a survey of their overall health and to determine whether P. alni was present in western Oregon riparian ecosystems. Over 1100 Phytophthora isolates were recovered, representing 20 species and 2 taxa. Phytophthora‐type cankers were observed in many trees, and their incidence was positively correlated with canopy dieback. High levels of mortality for red alder were not observed, which suggests these Phytophthora species are not aggressive pathogens. To test this hypothesis, three stem wound inoculations and one root dip were conducted on red alder seedlings using 13 Phytophthora species recovered from the riparian survey. Ten of the 13 Phytophthora species produced significant lesions in at least one pathogenicity test. Phytophthora siskiyouensis produced the largest lesions on red alder from the two stem wound inoculation tests conducted under summer conditions, while P. taxon Pgchlamydo caused the largest lesions during the winter stem wound inoculation test. Phytophthora gonapodyides, P. taxon Pgchlamydo and P. siskiyouensis have previously been found associated with necrotic alder roots and bole cankers in the field, and with the pathogenicity results reported here, we have established these species as causes of Phytophthora root disease and Phytophthora bole canker of alder in Oregon. While none of the Phytophthora species were especially aggressive towards red alder in the pathogenicity tests, they did cause localized disease symptoms. By weakening the root systems or boles of alders, the Phytophthoras could be leaving alders more susceptible to other insects and pathogens.     

Agrobacterium tumefaciens‐mediated transformation for targeted disruption and over expression of genes in the poplar pathogen Sphaerulina musiva

Sphaerulina musiva causes both leaf spots and cankers on poplar. Leaf spots can lead to defoliation and cankers on branches and primary stems can lead to stem breakage and tree mortality. The recent availability of both the S. musiva and Populus trichocarpa genomes offers a great opportunity to study host–pathogen interactions. To better understand the factors involved in S. musiva pathology, we present a strategy for the transformation of this species using Agrobacterium tumefaciens. Binary plasmids were generated with hygromycin B phosphotransferase (hph) flanked by upstream and downstream sequences of polyketide synthase‐like (PKS‐L1) gene to generate targeted gene disruptants by homologous recombination. Plasmids were also constructed for constitutive expression reporter genes eGFP and mCherry to help with histological characterization of the pathogen during infection. Gene knockouts were identified by PCR and confirmed by sequencing and Southern blotting. No significant differences were observed in melanin production between PKS‐L1 disruptants and wild type isolates. Colonies expressing reporter genes were identified by fluorescent stereomicroscopy. This method is a promising tool for the characterization of pathogen genes through reverse and forward genetics and for introducing markers for histopathological study.     

Phytophthora root and collar rot of mature Fraxinus excelsior in forest stands in Poland and Denmark

In recent years, Common ash (Fraxinus excelsior) throughout Europe has been severely impacted by a leaf and twig dieback caused by the hyphomycete Chalara fraxinea. The reasons for its current devastating outbreak, however, still remain unclear. Here, we report the presence of four Phytophthora taxa in declining ash stands in Poland and Denmark. Phytophthora cactorum, Phytophthora plurivora, Phytophthora taxon salixsoil and Phytophthora gonapodyides were isolated from rhizosphere soil samples and necrotic bark lesions on stems and roots of mature declining ash trees in four stands. The first three species proved to be aggressive to abscised roots, twigs and leaves of F. excelsior in inoculation experiments. Soil infestation tests also confirmed their pathogenicity towards fine and feeder roots of ash seedlings. Our results provide first evidence for an involvement of Phytophthora species as a contributing factor in current decline phenomena of F. excelsior across Europe. Specifically, they may act as a predisposing factor for trees subsequently infected by C. fraxinea. Phytophthora species from ash stands also proved to be aggressive towards a wide range of tree and shrub species commonly associated with F. excelsior in mixed stands. Although damage varied considerably depending on the Phytophthora species/isolate–host plant combination, these results show that many woody species may be a potential source for survival and inoculum build‐up of soilborne Phytophthora spp. in ash stands and forest ecosystems in general.     

Newly recognized bacteria involved in trunk scab and canker of white and hybrid poplars

In a 4‐year‐old collection of native Italian Populus alba, growing in the eastern Po valley, many trunks showed severe symptoms characterized by brown spots, similar to trunk scab and/or by cankers. Canker‐like growths, often with gall‐like‐formations in the earlier developmental phases were also found on some clones of P. trichocarpa and their hybrids, as well as on P. deltoides growing in several Northern Italian nurseries. Bacteria and fungi present in the affected tissues and potentially involved in the disease were examined. No fungi were detected, however, several species of bacteria were isolated and identified: Pseudomonas mendocina and Erwinia herbicola group, from the brown spots; Erwinia carotovora sub sp. carotovora and, occasionally, E. herbicola group, from cankers. Only E. carotovora sub sp. carotovora produced clear symptoms of canker when artificially inoculated on young plants of different poplar species. This is the first time this symptomology on poplar species has been clearly related to E. carotovora sub sp. carotovora.     

Response of planted beech (Fagus sylvatica L.) and Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) saplings to herbaceous and small shrubs control on clearcuts

The reaction of young beech (Fagus sylvatica L.) and Douglas fir (Pseudotsuga menziesii (Mirb.) Franco) saplings on competition of two types of vegetation—(1) gramineous with mainly Agrostis capillaries, Calamagrostis epigejos, Deschampsia flexuosa, and (2) small shrubs with mainly Rubus fruticosus and R. idaeus—on clear cuts on two sites was studied for 2 years. Half the sample saplings were released from competing vegetation by repeated herbicide applications. This treatment significantly raised the diameter increment in both species at the site with higher competition intensity, and more strongly after the removal of small shrubs than after the removal of grasses. Sapling length increment was not significantly affected. After being released from small shrubs, saplings of both species developed a smaller specific fine root length (cm g⁻¹ fine root biomass) than unreleased saplings during the second year which was characterized by low rainfall. Root nitrogen concentration significantly increased after weed control in both vegetation types. Sapling foliar content of main nutritional elements was negatively related to dry mass and total chemical content of surrounding ground vegetation. Based on these results, a release from ground vegetation could be a useful tool to improve growth of planted beech and Douglas-fir saplings on sites with well-developed small shrubs competition (mainly by Rubus fruticosus and R. idaeus), or under fairly dry conditions.     

Incidence of Gremmeniella abietina in planted seedlings of Picea abies and Pinus sylvestris in northern Finland

The ascomycete Gremmeniella abietina causes a disease (scleroderris canker) on conifers. The pathogen kills terminal shoots and branches of Pinus sylvestris and Picea abies, sporulates on host shoots and causes leader changes (i.e. dieback). Damage caused by G. abietina was investigated in young P. abies and P. sylvestris plantations in northern Finland. Side branches from seedlings of both species and the main stems of P. sylvestris were collected from three sites in two locations (Poksa 1 and 2, Kivalo) in northern Finland. The number of cankers and leader changes was counted in branches of each age (i.e. year) in a total of ca. 6300 shoots of P. abies and 1200 shoots of P. sylvestris. Cankers were common on both P. abies and P. sylvestris in Poksa 1 (2002–2008) and on P. abies in Poksa 2 (1998–2001). In Kivalo, cankers occurred only sporadically on P. sylvestris. Leader changes were most frequent in 1999–2009 in Poksa 1, in 2001–2009 in Poksa 2 and in 2003 in Kivalo. Cankers and other symptoms of infection were more obscure on P. abies than on P. sylvestris. According to both conidial morphology and molecular analysis, the strain of G. abietina infecting both P. abies and P. sylvestris was small‐tree type (STT). This is the first report of G. abietina, STT or B type, injuring P. abies in plantations.     

Beech decline in Central Europe driven by the interaction between Phytophthora infections and climatic extremes

During the past decade, and in particular after the wet year 2002 and the dry year 2003, an increasing number of trees and stands of European beech (Fagus sylvatica L.) in Bavaria were showing symptoms typical for Phytophthora diseases: increased transparency and crown dieback, small‐sized and often yellowish foliage, root and collar rot and aerial bleeding cankers up to stem heights of >20 m. Between 2003 and 2007 134 mature beech stands on a broad range of geological substrates were surveyed, and collar rot and aerial bleeding cankers were found in 116 (86.6%) stands. In most stands the majority of beech trees were declining and scattered or clustered mortality occurred. Bark and soil samples were taken from 314 trees in 112 stands, and 11 Phytophthora species were recovered from 253 trees (80.6%) in 104 stands (92.9%). The most frequent species were P. citricola, P. cambivora and P. cactorum. Primary Phytophthora lesions were soon infected by a series of secondary bark pathogens, including Nectria coccinea, and wood decay fungi. In addition, infected trees were often attacked by several bark and wood boring insects leading to rapid mortality. Bark necroses were examined for their probable age in order to determine whether the onset of the current Phytophthora epidemic was correlated to rainfall rates recorded at 22 Bavarian forest ecosystem monitoring stations. A small‐scale survey in nine Bavarian nurseries demonstrated regular infestations of all beech fields with the same range of Phytophthora species. The results indicate that (1) Phytophthora species are regularly associated with beech decline and may also be involved in the complex of ‘Beech Bark Disease’, (2) excessive rainfalls and droughts are triggering the disease, and (3) widespread Phytophthora infestations of nursery stock might endanger current and future silvicultural projects aiming on the replacement of non‐natural conifer stands by beech dominated mixed stands.