Armillaria, and the closely related genus Desarmillaria, are a diverse group of fungi with over 40 species. Many species of Armillaria cause Armillaria root rot in a wide range of hosts. This disease has caused severe losses in diverse ecosystems worldwide, in both natural and managed environments. Despite the considerable efforts invested in assessing the varying degrees of resistance among diverse woody hosts to Armillaria, most host screening studies have neglected the integration of concepts and mechanisms pertaining to woody host defense. While a modest body of knowledge exists concerning host defense mechanisms against Armillaria, the research is predominantly confined to selective host species within forest ecosystems. Thus, a multitude of theoretical and applied questions pertaining to the reactions and resistance of woody hosts to pathogenic Armillaria species continue to remain unanswered. The present review consolidates the current understanding surrounding defense mechanisms exhibited by diverse woody hosts in response to infection by pathogenic Armillaria species. Additionally, it outlines potential avenues for future research in this domain.

Armillaria root rot (ARR), caused by Armillaria species and Desarmillaria tabescens , is a severe disease that affects stone fruit trees in the United States. One strategy to mitigate the impact of this disease is to develop ARR-resistant rootstocks. However, current techniques to screen Prunus species for resistance to ARR are time-consuming, labor-intensive, and may not fully replicate field conditions. To address these limitations, we developed a new rapid in vitro screening assay, which uses roots of 2-year-old Prunus rootstock genotypes. We screened 12 Prunus genotypes against Armillaria mellea , Armillaria solidipes , and Desarmillaria tabescens in vitro. Freshly excavated root segments were placed next to or on top of fungal cultures. After 21 days, the circumferential percentage and horizontal length of the fungal colonization and the ability of the fungus to enter through root periderm were evaluated. The root tissue surrounding the infection was also evaluated to assess any response reactions against the ARR pathogens. Our results showed that inoculated root tissues displayed signs of fungal infection, and infection and host responses varied among the Prunus genotypes. Host responses similar to those observed in the field, such as compartmentalization of infected tissue with barrier zones, necrophylactic periderm formation, and callus formation on root surfaces, were observed and were more evident in less susceptible genotypes. In conclusion, our newly developed assay, which uses freshly excavated roots from 2-year-old rootstocks, can rapidly screen Prunus genotypes for resistance to ARR.

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.

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.

Variation in susceptibility of 28 Prunus rootstock genotypes to the causal agents of Armillaria root rot, Armillaria mellea , A. solidipes, and Desarmillaria tabescens , were studied by conducting in vitro root screening assays. Root segments with wounded and intact periderm were placed next to and on the top of the fungal cultures. At day 21, the percent success of fungal penetration and the circumferential and longitudinal lengths of fungal growth were measured. A parallel investigation using the inoculated root segments was carried out to characterize the active host defense mechanisms involved, including anatomical responses in bark and wood. Overall, the success of penetration and the longitudinal and circumferential spread of Armillaria spp. and D. tabescens were significantly different among various Prunus rootstock genotypes. None of the tested rootstock genotypes were completely resistant to infection. However, plum and plum derived rootstocks, and some of the cherry genotypes were less susceptible to infection compared to the peach genotypes. The host’s ability to limit infection by Armillaria spp. and D. tabescens was not limited to a single mechanism but appeared to be regulated by several collective nonspecific host responses acting together. Differential levels of a series of non-specific coordinated events were triggered in Prunus genotypes such as the formation of new callus tissue on the root surface, a colored reaction zone, necrophylactic periderm, new cells, and new vascular cambium to compartmentalize the pathogen. The host responses were elevated in the genotypes with low level of infection as compared to highly infected genotypes.

Armillaria mellea and A. solidipes are two of the primary causal agents of Armillaria root rot (ARR) in over 500 woody hosts. Although extensive research on this disease has been conducted over the past few decades, finding controls for ARR has been challenging. This is largely due to the lack of knowledge of the fungal infection and host response processes. It is important to understand the infection process in order to develop new strategies to control the disease. In this review, the initial steps of the infection process of A. mellea and A. solidipes are discussed, with focus on fungal attachment and penetration. A mucilaginous substance produced at the rhizomorph tip is thought to aid attachment of the pathogen to host. Toxins and cell wall degrading enzymes produced by the fungus together with mechanical pressure generated by the rhizomorph is speculated to support fungal penetration. However, a detailed description of the Armillaria spp. infection process is lacking in the literature, and this illustrates the need for further studies.

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.

We describe a holistic in vitro technique for inoculating roots of Prunus species with Armillaria solidipes, which is faster and more successful than previous methods. This method allows associated active and passive host defences to be assessed. Sterile root segments of three Prunus spp. were placed next to or on top of 14-day-old cultures of A. solidipes. At 21 day, the success of fungal penetration and length of the fungal colonization, and host responses were evaluated. Also, an anti-Armillaria and anti-Cladosporium activity detection assays were conducted by utilizing the root periderm, the first tissue that needs to be penetrated by fungus. These methods revealed the variation in the relative tolerance of three Prunus spp. to A. solidipes. The overall success of fungal colonization in the wounded and intact root, host defence, and antifungal activity significantly differed among three Prunus spp. Results indicate that this in vitro method can be used as a preliminary step in screening tree species to Armillaria spp.

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.

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.

Variation in the pathogenicity of 42 isolates of Leptographium terebrantis to Pinus taeda was examined. Stems of 1-year-old P. taeda seedlings were artificially inoculated with the fungal isolates. Eight weeks following inoculation, L. terebrantis isolates caused dark necrotic lesion and sapwood occlusion in the seedling stems. The fungal isolates varied in their ability to cause lesion and tissue occlusion. Lesions caused by fungal isolates were, however, significantly longer than the control. Results suggest that different isolates of L. terebrantis may not put the health of P. taeda at equal risk.

Pinus taeda (loblolly pine), a most widely cultivated timber species in the southern U.S., creates 110,000 job opportunities and contributes 30 billion dollars per year to the economy. However, insect-vectored root-infecting ophiostomatoid fungi, Leptographium terebrantis, and Grosmannia huntii are potential threats to sustainable P. taeda forest management in the southern U.S. Understanding the intra-species response of P. taeda to these fungi is critical to mitigate the potential problem due to these fungi. Thus, the objectives of my research are: (i) to determine the intraspecific tolerance/susceptibility of P. taeda to L. terebrantis and G. huntii, (ii) to understand whether intraspecific tolerance of P. taeda to L. terebrantis and G. huntii remain same regardless of the tree growth stage (iii) to understand the interaction of the vascularinhabiting fungi and P. taeda under drought conditions, (iv) to determine the antibiosis potential of these fungi by plant growth-promoting rhizobacteria (PGPR), (v) to understand whether PGPR can induce resistance of P. taeda families to these fungi, (vi) to determine the intraspecific variation in virulence of L. terebrantis, (vii) to determine the growth potential of most virulent L. terebrantis at different inoculum densities in P. taeda wood segments, and (viii) to determine the growth potential of various blue-stain fungi on P. taeda stem segments. In study 1, seedlings from 94 P. taeda families were artificially inoculated at the stem with L. terebrantis and G. huntii and family responses were studied. In study 2, the roots of the mature P. taeda trees from 4 families were inoculated with these two fungi to ii understand the intraspecific response of mature trees. In study 3, P. taeda families were exposed to drought and simultaneously inoculated with vascular-inhabiting fungi and impacts were studied. In study 4, PGPR strains and ophiostomatoid fungi were plated together in a dual agar plate and the antibiosis potential of PGPR strains to fungi was studied. In addition, induced systemic resistance of P. taeda to L. terebrantis and G. huntii were studied by inoculating PGPR in soil and fungi in stems of P. taeda seedlings. In study 5, most virulent L. terebrantis was inoculated to P. taeda stem segments at different inoculum densities. In study 6, various blue-staining ophiostomatoid fungi were cultured in P. taeda stem segments to study their growth potential. The results suggest P. taeda families vary in tolerance to ophiostomatoid fungi with potential for selection of relatively tolerant families. Moreover, this intra-species variation in tolerance is an inherent character of P. taeda, regardless of the tree growth stage. The growth and productivity of P. taeda seedlings decrease and fungal pathogenicity increase under severe drought. Specific strains of PGPR have the ability to inhibit the growth of blue-staining fungi in vitro. Specific PGPR strains have the capacity to induce systemic resistance of P. taeda during fungal infection. Fungal growth and blue-staining potential are high when fungal inoculation points are closer. Growth and staining potential of L. terebrantis in P. taeda stem segment is higher compared to G. huntii and G. alacris. This study provides meaningful insights into P. taeda and ophiostomatoid fungal interaction.