Intraspecies variation of mature Pinus taeda in response to root‐infecting ophiostomatoid fungi

Seedling screening studies have shown intraspecies variation in susceptibility of Pinus taeda (loblolly pine) to Leptographium terebrantis and Grosmannia huntii, the causal agents of root infection in Pinus species. However, it is critical to understand the susceptibility of mature P. taeda trees. Roots of mature P. taeda families determined as susceptible and tolerant to L. terebrantis and G. huntii by previous seedling screening trials were artificially inoculated with the same fungal isolates. Dark necrotic lesion and vascular occlusion were recorded 8 weeks later. Families previously considered as susceptible had relatively longer lesions and occlusions. The variation in susceptibility to the two fungi remained the same as exhibited by families in the seedling trial. These results suggest intraspecies variation in relative susceptibility of P. taeda to L. terebrantis and G. huntii remains similar regardless of the age of the tree.     

Variation in resistance of loblolly pine (Pinus taeda L.) families against Leptographium and Grosmannia root fungi

Pine decline poses a serious threat to forest sustainability in the south‐eastern United States. Complex interactions between biotic and abiotic factors are involved in the decline and include root‐feeding bark beetles and their associated fungal genera, Leptographium and Grosmannia. A study was conducted to determine the relative tolerance of loblolly pine (Pinus taeda L.) families when challenged with either Leptographium or Grosmannia species. In the first study, bare root seedlings from 23 loblolly pine families were screened with L. procerum, L. terebrantis, G. huntii and G. alacris using an artificial inoculation method. In a second study, containerized seedlings from 27 loblolly pine families were screened with G. huntii and L. terebrantis. Measured seedling responses to the inoculations included lesion length, lesion width and occlusion of vascular tissues, measured 8 weeks after inoculations. The most common host response was dark brown lesions and resinous occluded stem tissue. Seedling families had a wide range of host responses to the different Leptographium and Grosmannia species and showed that it could be possible to select the families that may be tolerant to Leptographium and Grosmannia fungal species based on these results.     

Pinus taeda L. response to differential inoculum density of Leptographium terebrantis colonized toothpicks

Bark beetle‐vectored ophiostomatoid fungi, Leptographium terebrantis, is inoculated on the roots and lower stems of stressed Pinus species during the feeding activity of bark beetle. To determine the exact host response following inoculation, it is critical to challenge the host with a realistic amount of fungal inoculum. Thus, we designed a series of stepwise experiments using L. terebrantis colonized toothpicks which focused on the inoculum transfer from the toothpicks to excised Pinus taeda stem segments and living saplings, respectively, at different inoculum densities. The toothpicks served as a substrate for fungal growth and sporulation and the inoculation showed their utility in eliciting host's response to the pathogen. The inoculated fungus caused blue‐stain and sapwood occlusions in P. taeda stems and saplings, respectively. The volume of occluded, visually damaged sapwood increased by 1.96 cm³ per radial inoculation point on average. Fungal colonized toothpicks can be used as a suitable alternative to agar discs for studying bark beetles vectored fungi and their host interactions.     

Root lesions in large loblolly pine (Pinus taeda L.) following inoculation with four root‐inhabiting ophiostomatoid fungi

Loblolly pine decline, characterized by deteriorating root systems leading to shortening and thinning of foliage, has been observed throughout portions of the south‐eastern United States. Several root‐inhabiting ophiostomatoid fungi, including Leptographium procerum, Leptographium terebrantis, Leptographium serpens, and Grosmannia huntii are associated with lateral root damage on declining loblolly pine. Trees of various ages were inoculated in primary lateral roots during fall (2006 and 2007) and spring (2007 and 2008). All fungi caused a darkened, resin‐filled lesion on the surface of the phloem, extending into the xylem that was larger than that of controls. Only lesions associated with G. huntii infection were significantly larger in the spring season, compared with the fall. Grosmannia huntii was found to be the most virulent fungus, causing lesions that were longer, deeper and larger than all other fungal species during the spring and larger than L. terebrantis and L. procerum in the fall. Leptographium serpens was the second most virulent fungal species, causing lesions larger than L. procerum and L. terebrantis (with the exception of lesion depth) during both seasons. These tests indicate that G. huntii and L. serpens are significant root pathogens, capable of causing considerable damage, while L. terebrantis and L. procerum may be less virulent. Depending on the actions of their vectors, G. huntii and L. serpens may be responsible for significant root deterioration and tree disease.     

Effects of oleoresins and monoterpenes on in vitro growth of fungi associated with pine decline in the Southern United States

As a means of exploring pine resistance to root disease and declines, the effects of host plant secondary metabolites on the growth of root colonizing fungi associated with three diseases/declines of southern pines – loblolly pine decline, littleleaf disease and annosum root rot were tested. The associated fungi –Leptographium huntii, L. serpens, L. terebrantis, L. procerum, Heterobasidion annosum and Phytophthora cinnamomi– were grown in saturated atmospheres or in direct contact with, pure monoterpenes and crude oleoresin collected from the four southern pines (Pinus taeda, P. eschinata, P. palustris and P. elliotti) for 7 day. Fungal growth was measured at 3, 5 and 7 day. Root‐infecting fungi differed significantly in sensitivity to crude oleoresin and pure monoterpenes. All fungi tested were inhibited, to some extent, by the resins tested. H. annosum and P. cinnamomi were strongly inhibited by all the monoterpenes tested. The ophiostomatoid fungi were significantly less affected by the compounds tested. L. huntii and L. serpens were less inhibited by monoterpenes than either L. terebrantis or L. procerum. These fungal growth studies show that the kind and amount of secondary metabolite produced by the host plant have a profound effect on tree pathogens. Alterations of tree physiology may have implications for defenses against tree pathogens as well as to the ecology and management of forest ecosystems. Difference in incidence of root disease observed in the field may be explained by the ability of the fungus to tolerate these host defense mechanisms.     

Variation in susceptibility of six pine species and hybrids to pitch canker caused by Fusarium circinatum

Six pine species or hybrids were tested for susceptibility to pitch canker caused by Fusarium  circinatum. Pinus  densiflora, Pinus  thunbergii, Pinus  x rigitaeda (Pinus  rigida × Pinus  taeda), P. rigida × P. x rigitaeda, Pinus  echinata and Pinus  virginiana were inoculated with three spore loads (50, 500 and 5000 per tree) of F. circinatum. External symptoms, lesion length, and the frequency of reisolation of the fungus were investigated. External symptoms were greatest in P. echinata, followed by P. virginiana, however, P. densiflora was not susceptible to F. circinatum. Based on mean lesion lengths, the six pine species or hybrids differed significantly (p < 0.01) in susceptibility to pitch canker. Pinus  echinata sustained the longest lesions, whereas P. densiflora sustained the shortest lesions. The effect of inoculum density was not significant among three spore treatments within species (p = 0.17), although lesion length was slightly greater at higher spore loads over all pine species. The fungus was reisolated from inoculated stems of all pine species tested, even on trees showing little or no damage from the disease. Additional studies are needed to further explore the basis for resistance to pitch canker.     

Susceptibility of longleaf pine roots to infection and damage by four root-inhabiting ophiostomatoid fungi

Restoration of longleaf pine-dominated uplands is common on many public and private lands throughout the southeastern United States. The once dominant longleaf pine ecosystem is important to many now-threatened and endangered plant and animal species, and land managers are increasing efforts to reestablish this fire-dependent forest. Unfortunately, tree mortality in longleaf pine has been observed following attempts to re-introduce prescribed fire. Root-inhabiting ophiostomatoid fungi and their insect vectors have invaded roots of symptomatic longleaf pine. Although, the relationship between ophiostomatoid fungi and longleaf pine roots is poorly understood. In order to assess the pathogenicity and virulence of four ophiostomatoid fungi to longleaf pine, trees within two broad age classes (20–30 and 40–60 years) were used for root inoculations during the fall of 2006 and 2007 along with the spring of 2007 and 2008. All fungal species consistently caused resin-filled, discolored lesions on the phloem surface extending to the xylem. The successful inoculation of healthy longleaf pine roots confirms the pathogenicity of Grosmannia huntii, Leptographium procerum, Leptographium serpens, and Leptographium terebrantis. G. huntii caused the largest lesions, including 22.20 cm², 13.37 cm², and 9.21 cm² larger than L. procerum, L. terebrantis, and L. serpens respectively. In contrast, L. procerum caused significantly smaller lesions than all other fungi, including 8.65 cm² smaller than L. terebrantis and 10.69 cm² smaller than L. serpens. Restoration efforts of longleaf pine may be affected by fungal root infection in the future. Future studies should focus on the interactions between stress factors associated with longleaf pine to define more clearly the ecological role of root-inhabiting ophiostomatoid fungi in the ecosystem.     

New hosts for Lecanosticta acicola and Dothistroma septosporum in newly established arboreta in Spain

A historical outbreak of needle blight disease was recorded during 2018 to 2019 in plantations of Pinus radiata and Pinus nigra in the North of Spain. The main pathogens involved in this historical outbreak were identified as Lecanosticta acicola and Dothistroma septosporum. Recently, a variety of tree species in three arboreta planted between 2011 and 2013 in the Basque Country as part of the European project REINFFORCE were showing symptoms of needle blight and defoliation. The aim of this study was to determine which pine species were affected with these pathogens. Tree species sampled included several provenances of P. brutia, P. elliottii, P. nigra, P. pinaster, P. pinea, P. ponderosa, P. sylvestris and P. taeda. Using molecular identification methods, Lecanosticta acicola was confirmed infecting Pinus brutia (Provenance: Alexandropolis, Greece and var. eldarica, Crimea) and represents a new host species for this pathogen. Pinus elliottii (Provenance: Georgia, USA) and P. ponderosa (Provenance: Central California, USA) are new host reports of L. acicola for Spain. Dothistroma septosporum was found for the first time on P. brutia (Provenance: Marmaris, Turkey) and P. ponderosa (Provenance: Oregon, USA) in Spain and was also detected infecting P. nigra (Provenance: Sologne Vayrières, France).     

Impacts of inoculation with Herpotrichia pinetorum,Gremmenia infestans and Gremmeniella abietina on Pinus nigra subsp. pallasiana and Cedrus libani seedlings in the field

Herpotrichia pinetorum, Gremmenia infestans and Gremmeniella abietina were inoculated onto 2‐year‐old Anatolian black pine (Pinus nigra subsp. pallasiana) and Taurus cedar (Cedrus libani) seedlings planted in a high mountain forest (1800 m a.s.l) in south‐western Turkey, to determine the effects of these fungi during winter. In June, 8 months after inoculation, 39.9% of experimental plants were dead and 20.4% of the surviving plants failed to flush. Gremmeniella abietina and H. pinetorum caused the most fatalities. Prevention of new shoot formation on surviving plants, however, was mainly an effect of G. abietina infections, although many surviving plants inoculated with G. infestans or H. pinetorum also failed to flush. All three pathogens had the potential to severely damage young plants of P. nigra subsp. pallasiana and C. libani growing at high elevations near to forests with heavy inoculum loads. The implications of this finding for P. nigra afforestations at high altitudes in Turkey are discussed. This study is also the first to report that G. infestans can infect and cause disease on young C. libani plants.     

Resistance levels of Spanish conifers against Fusarium circinatum and Diplodia pinea

Pitch canker, caused by Fusarium circinatum, and Diplodia shoot blight, caused by Diplodia pinea, are both damaging to pines (Pinus spp.) grown in plantations throughout the world, including Spain. To assess the potential for interspecific differences in susceptibility to contribute to the management of pitch canker and Diplodia shoot blight in the Atlantic region of Spain, the present study was undertaken to characterize the susceptibility of six pine species (P. sylvestris, P. nigra, P. pinaster, P. radiata, P. halepensis and P. pinea) and Douglas‐fir (Pseudotsuga menziesii) to F. circinatum and D. pinea. Based on inoculations of 2‐year‐old trees, Ps. menziesii, P. pinea and P. nigra were the most resistant to F. circinatum, with lesion lengths ranging from 3.7 to 21.5 mm, 2.2 to 12.6 mm and 2.8 to 30.9 mm, respectively. At the other extreme, Pinus radiata was the most susceptible, sustaining lesions that ranged from 8.5 to 74.8 mm in length. Pinus sylvestris, P. pinaster and P. halepensis showed an intermediate response to F. circinatum. Broadly similar results were observed in inoculations with D. pinea, with Ps. menziesii being relatively resistant and P. radiata being highly susceptible. Consistent with these results, field surveys revealed no pitch canker in stands of Ps. menziesii and low severity of Diplodia shoot blight, whereas P. radiata was severely affected by both diseases. Our findings suggest that selection of appropriate species can greatly reduce the risk of damage from two important canker diseases affecting pine plantations in the Atlantic region of Spain. Furthermore, intraspecific variation in susceptibility implies that selection may allow for the enhancement of resistance in otherwise susceptible species.     

Incidence of dieback disease following fungal inoculations of sexually and asexually propagated shisham (Dalbergia sissoo)

Shisham (Dalbergia sissoo) is an important multipurpose tree with great economic importance, but this tree has been devastated by dieback disease. Seedlings and asexually propagated (cuttings) plants were artificially inoculated with four fungi (Fusarium solani, Botryodiplodia theobromae, Curvularia lunata and Ganoderma lucidum) to evaluate the potential role of these fungi in shisham dieback disease. Results at 2 years revealed that highest disease was caused by inoculation of F. solani (31.39%), followed by B. theobromae (19.042%) and C. lunata (12.22%), but no dieback disease was caused by G. lucidum. During both years, seedlings exhibited greater susceptibility to disease (17.24%) compared to cuttings (7.83%). In particular, F. solani caused more disease in seedlings (46.18%) compared to cuttings (16.61%). With the F. solani inoculations, maximum disease rate was observed at 8 weeks post‐inoculation both in seedlings (77%) and in cuttings (31%), but the maximum disease increase was observed at 4–5 weeks post‐inoculation. Analysis of variance showed significant differences among the different fungi and also between seedlings and cuttings. F. solani can be considered as a major fungal pathogen contributing to dieback disease of shisham, and asexual propagation can reduce the severity of dieback.     

Pathogenicity of ophiostomatoid fungi on Picea abies in Slovenia

Pathogenic fungi can survive and develop in living plants, often causing diseases in the host. Some theories speculate that pathogenic ophiostomatoid fungi provide benefits to its vectors – bark beetles – by overcoming the tree's defence mechanisms. This study reports the results of an experiment in south‐eastern Europe in which mature and seedling Norway spruce trees were artificially inoculated with various ophiostomatoid fungi. The aim of the experiment was to determine the relative virulence of ophiostomatoid fungi by assessing the ability of the fungi to stimulate host tree defence mechanisms through inoculation experiments. Experiments were performed by inoculation of Picea abies in seedling and mature trees. The following fungi were used in low‐density and seedling inoculations: Ophiostoma ainoae, O. brunneo‐ciliatum, Grosmannia cucullata and an unidentified Leptographium sp., O. bicolor, O. fuscum, O. piceae, G. penicillata and G. piceiperda. Endoconidiophora polonica was used in mass and seedling inoculations. Various characteristics such as host vitality, blue stain, lesion and resin outflow were measured before and after the trees were felled. E. polonica caused blue stain, induced large lesions and killed some of the mature trees and seedlings, confirming earlier reports that it is a strong wound pathogen. Only E. polonica, Leptographium sp. and O. ainoae caused blue stains in the sapwood of inoculated seedlings. In low‐density inoculations, G. piceiperda induced intense necrosis and had higher values for all the characteristics monitored. Some of the other ophiostomatoid fungi showed a moderate level of pathogenicity. Fungi with the capacity to stimulate a host defence mechanism could play a role in the establishment of bark beetle populations.     

Tolerance of Pinus patula full-sib families to Fusarium circinatum in a greenhouse study

The pitch canker fungus, Fusarium circinatum, has caused large-scale mortality of young Pinus patula Schiede and Deppe ex Schltdl. and Cham. seedlings in nurseries in South Africa since 1990. Diseased seedlings have been inadvertently carried to the field, which in turn have died and has reduced stocking below an acceptable level. Tree breeders have suggested that the only long-term solution to limit infection by this pathogen is to identify and deploy tolerant P. patula families. A commonly used technique to identify tolerant clones is to artificially inoculate open-pollinated progeny from orchard clones with F. circinatum under greenhouse conditions. In these trials, large variation in tolerance to the pathogen among seedlings within open-pollinated families was observed and this could be influenced by the pollen parent. Therefore, identifying individual full-sib families, where both parents are known, should improve the identification of tolerant families, which can then be repeated. In this study, cuttings from control-pollinated P. patula seedling hedges were inoculated with F. circinatum in a greenhouse. The results showed large family variation where some of the full-sib families were similar in tolerance to P. elliottii Engelm. var. elliottii seedlings. Therefore, it is recommended that breeders focus on identifying specific family combinations that are more tolerant to F. circinatum.     

Infection approach of Diplodia sapinea,the causal agent of tip blight of Pinus tabulaeformis in China

To clarify the infection approach of Diplodia sapinea, a pathogen that causes tip blight of Pinus tabulaeformis, the infection process of the pathogen in needles was observed using scanning electron microscopy (SEM). In addition, the disease incidence on branches damaged by Aphrophora flavipes (Hemiptera: Cercopidae) and Dioryctria splendidella (Lepidoptera: Pyralidae) in the forest was also investigated. Then, branches and needles of P. tabulaeformis were inoculated using the D. sapinea spore suspension under indoor and field conditions. The results showed that the damage caused by A. flavipes could aggravate the occurrence of tip blight of P. tabulaeformis to some extent. Moreover, the pathogen could also penetrate 1‐, 2‐ and 3‐year‐old pine needles through stomata in the field. The pathogen infected the 1‐year‐old branches first and then gradually spread to 2‐ and 3‐year‐old branches.     

Host specificity of co‐infecting Botryosphaeriaceae on ornamental and forest trees in the Western Balkans

The Botryosphaeriaceae is a diverse family of endophytes and fungal pathogens of mainly woody plants. We considered the host range and distribution of these fungi by sampling diseased ornamental and forest trees and shrubs in Serbia, Montenegro, Bosnia and Herzegovina, spanning a Mediterranean and a Continental climatic region. In total, ten Botryosphaeriaceae species were identified in the Western Balkans and with the exception of Sphaeropsis visci and Phaeobotryon cupressi, which occurred on one host, all the species had a broader host range. Phaeobotryon cupressi was found only in the Mediterranean region and S. visci, Dothiorella sp., Dothiorella sarmentorum and Diplodia seriata were present only in the Continental region. Pathogenicity tests were conducted on a variety of hosts from which the Botryosphaeriaceae species were isolated. These included leaves and/or stems of seedlings of 21 hosts, and cut leaves and/or branches of six hosts. Moreover, stems of seedlings of Chamaecyparis lawsoniana, Cedrus deodara, Picea omorika, Pinus patula and Eucalyptus grandis were inoculated as hosts from which some or all of the Botryosphaeriaceae species used for inoculation were not isolated. Inoculations showed that the majority of these fungi could also co‐infect hosts other than those from which they were isolated. The results suggest that most of the species have broad host ranges and can potentially cause disease on a broad range of tree species under certain conditions.     

Early genetic evaluation methods for cyclic growth in Pinus taeda L.

The seedling growth trait, cyclic growth, is well-correlated with older-tree heights measured in Pinus taeda L. field trials. These results have been repeatable across locations and seed sources if a standard greenhouse procedure is followed. This work is been limited to cyclic growth measured on P. taeda families and seed sources. The use of a standard greenhouse procedure will facilitate comparison with other early genetic evaulation methods for P. taeda and with similar studies on other Pinus species.     

Growth and wood density of Pinus taeda L. as affected by shelterwood harvest in a two-aged stand in Southern Brazil

Pine plantations are an important wood source in Brazil, with Pinus taeda being most frequently planted. Most pinewood is directed to the paper and pulp industry, but there is an increasing demand for wood for solid end-uses, requiring large stems from longer rotations which can be obtained using P. taeda as the canopy in two-aged stands. We evaluated radial growth and wood density at different stem heights of P. taeda in the highlands of Southern Brazil over a production period of 36 years and subjected to shelterwood harvest. Cross-sectional disks were obtained from 15 trees in different stem heights; 10 were used for growth analyses and 5 for growth and density analyses. We used disk images and X-ray techniques for growth and density analyses, respectively. Samples were analyzed for ring (width and density), earlywood, and latewood (width, density, and proportion). Ring width varied between 0.4 and 1.7 cm, with the widest rings in the first years (3–5 years.) of growth. Ring density increased with age, with higher densities on the lower stem portions. Mature wood started to be formed from the 16th ring onwards. Shelterwood harvest affected both ring width and density, but the effects on ring width lasted for at least 5 years, while the effects on wood density were short-lasting. Mature P. taeda trees increased their size after the shelterwood harvest without compromising their wood density. Longer production periods of P. taeda as retained trees in the canopy of two-aged stands provide high-quality wood for structural purposes.

     

Growth rates of Heterobasidion annosum s.s. and H. parviporum in functional sapwood of Pinus sylvestris and Picea abies

Growth rates of H. annosum s.s. and H. parviporum were investigated in the functional sapwood of young Pinus sylvestris and Picea abies plants as an indicator of the relative susceptibilities of the hosts to these pathogens. The stems of 520 five‐year‐old P. abies and 321 four‐year‐old P. sylvestris plants were inoculated and the extent of infection determined 16 weeks later. H. annosum s.s. grew further than H. parviporum in P. sylvestris sapwood, while in P. abies, no differences between the two Heterobasidion spp. were found. Both H. annosum s.s. and H. parviporum spread faster in the sapwood of P. abies than in P. sylvestris. There was high within‐host species variation in growth rates for both P. sylvestris and P. abies suggesting it may be possible to identify tree genotypes with lower susceptibility.     

Pathogenicity of Heterobasidion annosum (Fr.) Bref. sensu stricto on coniferous tree species in Turkey

Two‐year‐old seedlings of Pinus brutia, P. brutia var. eldarica, Pinus pinea and 3‐year‐old seedlings of Pinus radiata, Pinus sylvestris, Pinus nigra and Cedrus libani were inoculated on the lower stem with isolates of Heterobasidion annosum s.s. collected from the Black Sea and Mediterranean regions of Turkey. In total, 315 seedlings were inoculated in April 2014 and incubated in a growth chamber for 7 weeks at 18–20°C. All isolates were pathogenic on the seven different hosts and had the ability to grow in living sapwood. The isolates had a greater growth on C. libani, P. sylvestris and P. radiata seedlings compared to plants of the other species tested. The least affected species were P. brutia and P. nigra. The isolates originating from the Black Sea region caused longer lesions on the hosts. Overall mortality during 7 weeks of incubation was 4%.     

Environmentally induced changes in the distribution of disease susceptibility phenotypes in Pinus radiata

Pinus radiata (Monterey pine) is highly susceptible to Fusarium circinatum, the cause of pitch canker, but heritable variation in resistance to this disease has been documented in P. radiata. In this study, the distribution of susceptibility phenotypes (=lesion lengths) was assessed by inoculating P. radiata trees with a spore suspension of F. circinatum. The results show the distribution of susceptibility phenotypes in naïve seedlings to be normally distributed, both in a breeding population and in a population established from seed collected in a native forest. This pattern aligns with expectations for a quantitatively inherited trait. However, after 2.5 years of growth in a forest, variation in susceptibility is no longer normally distributed but rather is positively skewed. Thus, exposure to the forest environment results in a distribution that is enriched for relatively resistant individuals. A similarly skewed distribution is observed in standing trees in a native population of P. radiata. The observed change in distribution cannot be attributed to ontological effects on resistance to pitch canker, because tree age accounts for almost none of the variation in susceptibility. We hypothesize that the change in distribution of susceptibility phenotypes is due primarily to infections and infection attempts by the microbiota resident in the forest.