High-quality fungal reference genomes are essential for comparative, functional and evolutionary studies, yet fungal genome features such as repeats, structural rearrangements, accessory chromosomes and intron-rich genes can complicate genome assembly and the selection of cost-effective sequencing strategies. Here, we benchmark fungal genome assembly performance using simulated and empirical short- and long-reads datasets to evaluate how sequencing depth, assembler choice and genome characteristics influence contiguity, completeness, accuracy and computational requirements. Using simulated reads from complete fungal genomes spanning diverse sizes and compositions, we evaluated short-reads (SR), long-reads (LR), hybrid and polished LR assemblies across sequencing depths from 10X to 100X. Key trends were validated using empirical sequencing data from 10 fungal isolates assembled with multiple strategies, including different Flye assembler parameter sensitivity and SR polishing. Across datasets, LR produced the largest improvements in contiguity, with most gains achieved at ~20-40X coverage and diminishing returns beyond moderate depth. SR polishing substantially improved base-level accuracy at relatively low cost, with ~10-20X coverage often sufficient to approach maximal error reduction. Hybrid assemblers showed strong algorithmic variability, with trade-offs between contiguity, error rates and computational demand. Genome architecture also influenced outcomes, as larger and more feature-dense genomes benefited more from long-read data while GC content had limited impact. Overall, our results suggest that moderate long-read coverage (~30-40X) combined with modest short-read polishing (~10-20X), particularly using Flye plus Polypolish, provides a strong balance of contiguity, completeness, accuracy and resource efficiency for generating high-quality fungal genome assemblies.

The influence of Leptographium terebrantis stem infection on constitutive and induced oleoresin and soluble phenolics in terminal shoots was assessed in a Pinus taeda plantation using three inoculum densities. The pathogen causes wilt disease by invading and colonizing root xylem tissues. We observed three levels of P. taeda defense in response to L. terebrantis during a two-year period interrupted by drought. Normal carbon allocation to growth was maintained and constitutive oleoresin production was observed at low and medium inoculum densities, with oleoresin induction observed among the medium inoculum density trees. At the high inoculum level, oleoresin flow occurred at a level similar to the medium inoculum density trees and carbon allocation to growth was diminished. Terminal shoot phenolic concentration was unaffected by the pathogen. Our findings validate the view that simultaneous occurrence of wilt disease and drought reduces constitutive and induced defenses by interfering with carbon fixation and allocation to growth.

The timber industry in the southeastern United States relies heavily on Pinus taeda. Over the past decade, impacts from Lecanosticta acicola causing Brown Spot Needle Blight have been increasing on P. taeda. Disease spread is well documented for living needles, with conidia spreading locally and ascospores spreading long distances. Still, little is known about survival of L. acicola in needles once removed from the tree. The objective of this study was to understand temporal persistence and seasonal sporulation patterns of L. acicola after being removed from the tree. Understanding how detached needles contribute to inoculum load is essential for better management practices, as some needles fall to the ground and others get stuck in the canopy, close to living foliage. In this study, infected needles were removed from living trees in November 2023 and March 2024 and the needles were monitored for 1 year after removal. Monitoring ended after 1 year due to annual needle fall, which adds fresh fallen needles. Each week, needles were selected and placed in sporulation chambers for identification. The microbe of interest, L. acicola, was recovered at the 1-year mark. Other microbes found were Pestalotiopsis sp., Diplodia sp., Hendersonia sp., Coleosporium sp., Lophodermium sp., Alternaria sp., and Trichoderma sp., with all but Coleosporium sp. and Lophodermium sp. recovered at the 1-year mark. These findings confirmed detached needles can continue to sporulate L. acicola for at least 1 year after removal. Future work should focus on determining L. acicola spread from detached needles, including both needles that get stuck in the canopy and needles that fall to the ground.

Pine forests are increasingly threatened by needle diseases, including Brown Spot Needle Blight (BSNB), caused by Lecanosticta acicola. BSNB leads to needle loss, reduced growth, significant tree mortality, and disruptions in global timber production. Due to its severity, L. acicola is designated as a quarantine pathogen in several countries, requiring effective early detection and control of its spread. Remote sensing (RS) technologies provide scalable and efficient solutions for broad-scale disease surveillance. This study systematically reviews RS-based methods for detecting BSNB symptoms, assessing current research trends and potential applications. A comprehensive bibliometric analysis using the Web of Science database indicated that direct RS applications for BSNB remain scarce. However, studies on other needle diseases demonstrated the effectiveness of multisource RS techniques for symptom detection, spatial mapping, and severity assessment. Advancements in machine learning (ML) and deep learning (DL) have further improved RS capabilities for automated disease classification and predictive modeling in forest health monitoring. Climate-driven factors, such as temperature and precipitation, regulate the distribution and severity of emerging pathogens. Geospatial analyses and species distribution modeling (SDM) have been successfully applied to predict BSNB pathogen’s range expansion under changing climatic conditions. Integrating these models with RS-based monitoring enhances early detection and risk assessment. However, despite these advancements, direct RS applications for BSNB detection remain limited. This review identifies key knowledge gaps and highlights the need for further research to optimize RS-based methodologies, refine predictive models, and develop early warning systems for improved forest management.

The severity of plant diseases caused by phytopathogenic fungi is shaped by both molecular-level virulence factors, ecological infection strategies. This study quantified the variances in disease severity driven by these predictors. To achieve this, we compiled data from experimental studies that compared disease outcomes between wild-type fungal strains and knock out mutants lacking genes suspected to influence pathogenicity. We used Cohen's d to quantify the effect size between measures of disease severity in these wild-type and mutant pairs and a ridge regression to quantify associations between pathogen traits and effect size. For predictor of disease severity, we found that knock outs of secreted enzymes resulted in smaller disease effect in wild-type strain relative to the mutant strain (scaled estimate = -5.780, p < 0.001), indicating potential redundancy or compensatory effects from other class of enzymes. However, when effector proteins were knocked out, the wild-type strains had significantly higher disease effect compared to the mutant strains (scaled estimate = 7.514, p < 0.001). Furthermore, necrotrophic fungi (scaled estimate = -6.009,p < 0.001) and those targeting roots and vascular tissues (scaled estimate = -2.510, p = 0.004) resulted in smaller disease effects in the wild-type compared to the knockout. In contrast, waterborne fungi (scaled estimate = 5.060, p < 0.001) and those targeting leaves and stems (scaled estimate = 2.656, p = 0.003) exhibited larger disease effects in the wild-type. Combined, our findings suggest that the influence of gene knockouts on disease severity is context-dependent, shaped by fungal lifestyle and infection strategy. This underscores the importance of integrating molecular data with ecological context when predicting or managing plant disease. These results can inform breeding programs and disease management strategies by identifying molecular targets likely to yield robust reductions in pathogenicity.

Brown Spot Needle Blight (BSNB) poses a significant threat to loblolly pine ( Pinus taeda ) forests in the southeastern United States, reducing timber yields, biodiversity, and overall forest health. Traditional detection methods rely on field-based assessments, which are time-intensive and impractical for large areas. Unmanned aerial vehicle (UAV)-based multispectral imaging presents a potential alternative, but its effectiveness for BSNB detection remains largely unexplored. To address this gap, we developed a UAV-based remote sensing framework to detect and map BSNB severity using multispectral imagery and machine learning. Specifically, we aimed to (i) classify and map BSNB severity using Support Vector Machines (SVM) and Artificial Neural Networks (ANN) and (ii) quantify the density of healthy and BSNB-infected trees using point cloud-derived metrics from UAV-based Structure from Motion (SfM). Field-based assessments across fourteen loblolly pine-dominated sites in the state of Alabama, provided BSNB-verified observations for model training and testing, and high-resolution UAV-based multispectral imagery were acquired using a DJI Mavic 3M. Spectral analysis of processed image bands and derived indices identified the Normalized Difference Vegetation Index (NDVI) and Soil-Adjusted Vegetation Index (SAVI) as optimal predictors of BSNB presence. Classification models achieved high accuracy, with SVM and ANN reaching 94.79% and 94.00% accuracy in Washington County, and 94.94% and 94.89% in Cullman County, respectively. Kappa coefficients ranged from 0.80 to 0.92 for SVM and 0.80 to 0.89 for ANN. This study provides one of the first systematic UAV-based approaches for BSNB detection and severity mapping, demonstrating the potential of combining multispectral imagery, SfM-derived metrics, and machine learning for operational forest health assessments.

A study was initiated in 2010 to evaluate the influence of cogongrass presence on soil and foliar contents of mid rotation loblolly pine stands in the Coastal Plain of Mississippi. Total carbon (TC) and total nitrogen (TN) soil stocks were higher in the presence of cogongrass (CO) compared with cogongrass free (NCO) stands. Significant differences were detected for TC and TN in all sampled soil layers. Treatment (TRT) (CO versus NCO) and soil depth (DPTH) significantly influenced total base cation content (TBC), most likely, due to the higher quantity of calcium (Ca) throughout the sampled profile. The interaction of TRT x DPTH was significant only for potassium (K). The most dominant cation on the exchange complex was aluminum (Al) with higher levels noted in NCO. Soil pHH2O levels were typical of loblolly pine stands with slightly higher levels noted for CO. Phosphorus (P) levels were considered insufficient for both CO and NCO but significant differences were noted in the immediate subsoil layer. Manganese (Mn) levels were higher in CO in all soil layers with significant differences in the surface and lowest sampled subsoil layer. Boron (B) was below detection levels in NCO and might be indicative of insufficiency. Soil physical conditions were more favorable to CO in the upper 20 cm but appeared to favor NCO below 30 cm. The presence of CO may have contributed to lower incremental growth and root proliferation of loblolly pine. Foliar contents were considered insufficient for P, sulfur (S), and copper (Cu) while Mn and B were in excess, especially Mn. The interaction of treatment (CO vs. NCO) x needle flush (FL) was significant for all nutrients except P, Mg, and Na.

The course of the bark beetle-vectored fungus, Leptographium terebrantis S. J. Barras and T. J. Perry, in stemwood growth loss of declining pines in the southeastern United States was assessed in a 13-year-old loblolly pine (Pinus taeda L.) plantation near Eufaula, Alabama, U.S.A. Using stem inoculation as a surrogate for root infection, we hypothesized that L. terebrantis infection impairs sapwood function and thus limits the tree leaf area (AL), new root production, and stemwood growth. Sterile toothpicks colonized by L. terebrantis at varying inoculum densities was used to elicit host growth responses. In the third year after inoculation, the root pathogen reduced the foliage moisture content, whole-tree leaf area (AL), the ratio of AL to tree sapwood area (AS), and stemwood growth in trees receiving the high inoculation treatment relative to those receiving the low or medium inoculation treatments, or the wound or control treatments after seven months of water deficit. The absence of a similar response to water deficit among trees that were noninoculated, wounded, or inoculated at the low or medium densities suggests that, in the loblolly pine–L. terebrantis pathosystem at our study site, the physiological stress caused by water deficit and the high inoculum density was required for the pathogen to elicit a stemwood growth loss. Thus, in loblolly pine forests of the southeastern United States, where climate and soil conditions yield prolonged periods of physiological stress, the presence of L. terebrantis has the potential to reduce stand volume and widen the gap between the predicted and actual stemwood production.

Leptographium terebrantis is an opportunistic root pathogen that has been implicated as a contributing factor of Pinus taeda decline and mortality over the past several decades in central parts of Alabama and Georgia, USA. We assessed the potential of L. terebrantis to initiate crown thinning in young P. taeda trees and hypothesized that L. terebrantis infestation will impose moisture stress on foliage, induce premature senescence, and cause loss of foliage biomass. The study was undertaken in a naturally regenerated 5-7-year-old P. taeda stand at Andalusia, Alabama in a completely randomized design using artificial inoculations of L. terebrantis colonized toothpicks. After four years of infestation, the pathogen caused sapwood occlusions and loss of sapwood function but failed to impose moisture stress on needles to induce premature senescence and loss of biomass. The new sapwood formed around the occluded area was devoid of pathogen infestation. The new growth was approximately twice the size of occluded tissue and compensated for the loss of old sapwood function to sustain tree growth. Results demonstrated that young P. taeda trees can tolerate L. terebrantis infection when stand conditions sustain the formation of new sapwood.

Leptographium terebrantis has been implicated as a contributing factor of P. taeda decline and mortality over the past several decades. We examined the potential of L. terebrantis to cause decline symptoms and determined the relationship between pathogen spread and the formation of new sapwood. The study was undertaken in a 13-y-old P. taeda plantation using artificial inoculations of fungal-colonized, sterilized toothpicks. We found that L. terebrantis was not only re-isolated from dying inoculated trees but caused decline symptomology and mortality at a high inoculum density. It was found that 20% mortality and severe growth loss among surviving trees occurred with L. terebrantis infection at the high density. At lower inoculum densities, trees produced a complete ring of new sapwood that appeared to sustain tree physiology. This suggests that management practices in P. taeda plantations which minimize bark beetle infestation and pathogen inoculum densities allow adequate sapwood function for sustained growth. ? 2021 Elsevier Ltd and British Mycological Society. All rights reserved.

Bark beetles and root weevils can impact forests through tree death on landscape scales. Recently, subterranean termites have been linked to these beetles via the presence of bluestain fungi (Ascomycota: Ophiostomataceae), which are vectored to trees by beetles. However, only a small subset of bluestain species have been examined. Here, we tested whether termite-bluestain association patterns in the field reflect termite feeding preference in laboratory choice trials. We documented the presence of four bluestain fungi (Leptographium procerum (W.B. Kendr.), L. terebrantis (Barras & Perry), Grosmannia huntii (Rob.-Jeffr.), and G. alacris (T.A. Duong, Z.W. de Beer & M.J. Wingf.) in the roots of 2,350 loblolly pine trees in the southeastern United States and whether termites were present or absent on these roots and paired this with laboratory choice feeding trials. Termites were found 2.5-fold on tree roots with at least one bluestain fungus present than tree roots without bluestain fungi. Although termites in this study and others were associated with L. procerum, L. terebrantis, and marginally G. huntii, termites only showed preferential feeding on wood inoculated with G. huntii in laboratory trials. This suggests that increased termite presence on wood with bluestain fungi may be driven by factors other than increased wood palatability. Termites could thus disproportionately affect wood turnover rates for specific pools (e.g., bark beetle and root weevil attacked trees) and in some cases (e.g., G. huntii) accelerate wood decomposition. This study supports the growing evidence that the association between subterranean termites and bluestain fungi is spatially and taxonomically widespread.

Ophiostomatoid fungi, Leptographium terebrantis and Grosmannia huntii, are among the most important agents of wood blue stain and root disease of Pinus species. A number of physical, chemical, and cultural strategies adopted to minimise the impact of these fungi are of limited success. Biological control of tree diseases with plant growth-promoting rhizobacteria (PGPR) is attractive given that they provide an alternative and supplement to synthetic chemicals without negative impact on the environment. While a significant number of studies have shown the ability of PGPR to control pathogens of agricultural plants, studies to understand the activity of PGPR against ophiostomatoid fungal pathogens of conifers is still lacking. To understand the utility of PGPR in treating blue stain in woods, twenty-seven Bacillus velezensis strains and single strains of Paenibacillus peoriae and B. altitudinis that had previously shown antibiosis against plant pathogens from six different genera were tested for in vitro antibiosis against L. terebrantis and G. huntii. To understand the efficacy of PGPR in controlling root-disease, Bacillus pumilus SE-34 and INR7, and Serratia marcescens (90-166), previously reported to have efficacy against a root pathogen in conifer, were tested for their capacity to induce resistance of P. taeda to these fungi. All PGPR strains inhibited the growth of L. terebrantis and G. huntii in vitro. In a second experiment, specific PGPR treatment resulted in increased seedling dry matter biomass and reduction of diseased tissue. The results demonstrate tested PGPR strains may have potential as biocontrol agents to the tested ophiostomatoid fungi.

Young Pinus taeda trees may tolerate Leptographium terebrantis infection when stand conditions support new sapwood growth devoid of pathogen-induced occlusion. Leptographium terebrantis S. J. Barras and T. J. Perry is an opportunistic root pathogen that compromises the xylem function of infected trees and is commonly associated with Pinus taeda L. stands that experience an unexplained loss of vigor in the southeastern U.S. To understand the relationship between L. terebrantis inoculation density, sapwood occlusion, and sapwood function characterized by hydraulic conductivity and moisture content, an artificial inoculation study was conducted in young P. taeda trees in a naturally regenerated stand over a 24-week period in south central Alabama. Four levels of increasing stem inoculation were used as a surrogate for comparable levels of woody root inoculation followed by an evaluation of pathogen-induced occlusion, sapwood function, and fascicle physiology. Occlusion of old sapwood intensified as L. terebrantis inoculum density increased, but occlusion was absent in current-year sapwood. Occlusion reduced sapwood hydraulic conductivity and moisture content but did not interfere with stomatal conductance. The vertical spread of L. terebrantis was correlated with losses of sapwood hydraulic conductivity and moisture content due to occlusion. Results demonstrate that the sapwood function of P. taeda is tolerant of the pathogen vascular occlusion when stand conditions sustain adequate carbon fixation for occlusion-free stemwood growth.

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 cm(3) 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.

Pinus taeda (loblolly pine) is one of the ecologically and economically important conifer species in the southeastern USA. However, a disease decline syndrome, southern pine decline (SPD), associated with beetle-vectored root-infecting fungi, has emerged as one of the major challenges confronting loblolly pine production in this part of the country. Although several studies have been conducted to screen the susceptibility of the commercially grown families to these fungi, little information exists on wood properties associated with the susceptible and resistant families. Thus, the objectives of this study were (1) to understand variation in wood quality parameters among the families regarded as susceptible and tolerant to SPD, (2) to evaluate the utility of acoustic tool to differentiate between those families. The results indicated the velocity, fiber length, microfibril angle and slenderness of the susceptible families are comparable or superior to those of the tolerant families. The mean error rate of classification associated with acoustic tool ranged from 35 to 40% depending on the distance between the transmitter and receiver probes. The mean error rate of classification was 35% when probes were placed 120 cm apart. The results from this study signify that a family tolerant to pine decline is not synonymous to a quality wood family and possibility of using acoustic tools to allocate pine species into PD susceptibility classes.

The objective of this study was to evaluate responses of containerized and bareroot seedlings from the same Pinus taeda L. families to ophiostomatoid fungi, Leptographium trerebrantis and Grosmannia huntii. Seedlings from four families were artificially inoculated with L. terebrantis and G. huntii. After 8 weeks, tissue necrosis and occlusion caused by the fungi were measured. Seedlings from both P. taeda stockypes showed similar susceptibility to fungi, suggesting both seedling stocktypes can be used to screen the susceptibility of P. taeda families against tested ophiostomatoid fungi. This paper was presented at the Joint Annual Meeting of the Southern Forest Nursery Association and the Northeast Forest and Conservation Nursery Association (Pensacola, FL, July 17–19, 2018).

We examined intraspecific and inter-year variation in tolerance of Pinus taeda to two ophiostomatoid fungi, Leptographium terebrantis and Grosmannia huntii. Containerized seedlings of P. taeda from 27, 32, 17 and 23 different elite genetic families were artificially inoculated with L. terebrantis and G. huntii in years 2013, 2014, 2016 and 2017, respectively. Six connector families were inoculated every year. Eight weeks post-inoculation, lesion and occlusion were measured on each seedling to determine the relative susceptibility/tolerance of families to these fungi. Pinus taeda families widely differed in these parameters suggesting intraspecific variation in the susceptibility/tolerance to the inoculated pathogens. The overall tolerance of the connector families to these fungi varied among the experimental years. These results showed that intraspecific variation to L. terebrantis and G. huntii exists among P. taeda families and it could be possible to select tolerant families to minimize the potential impact due to these fungi.

Summary 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.

The complex interaction of various biotic and abiotic factors may put the overall stand health ofPinusspp. at risk. A study was designed to determine the combined impact of drought and vascular-inhabiting fungi (Leptographium terebrantisandGrosmannia huntii) inPinus taeda. Seedlings from twoP. taedafamilies were planted and watering treatments, (i) normal watering, (ii) moderate drought, and (iii) severe drought, were applied. One month following the initiation of watering treatments, seedling stems were artificially inoculated withL. terebrantisandG. huntii. Drought and fungal interaction significantly affected lesion length/seedling height, occlusion length/seedling height, and seedling fine root biomass.Leptographium terebrantiswas more pathogenic under moderate and severe drought than normal watering condition, whereas the pathogenicity ofG. huntiiremains unaltered. The susceptibility of the families to vascular-inhabiting fungi remained the same under different watering treatments. Drought and specific vascular-inhabiting fungi may negatively impactP. taedastand health.