New insights into the role of constitutive bacterial rhizobiome and phenolic compounds in two Pinus spp. with contrasting susceptibility to pine pitch canker

The rhizobiome is being increasingly acknowledged as a key player in plant health and breeding strategies. The pine pitch canker (PPC), caused by the fungus Fusarium circinatum, affects pine species with varying susceptibility degrees. Our aims were to explore the bacterial rhizobiome of a susceptible (Pinus radiata) and a resistant (Pinus pinea) species together with other physiological traits, and to analyze shifts upon F. circinatum inoculation. Pinus seedlings were stem inoculated with F. circinatum spores and needle gas exchange and antioxidant-related parameters were analyzed in non-inoculated and inoculated plants. Rhizobiome structure was evaluated through 16S rRNA gene massive parallel sequencing. Species (non-inoculated plants) harbored distinct rhizobiomes (<40% similarity), where P. pinea displayed a rhizobiome with increased abundance of taxa described in suppressive soils, displaying plant-growth-promoting (PGP) traits and/or antifungal activity. Plants of this species also displayed higher levels of phenolic compounds. Fusarium circinatum induced slight changes in the rhizobiome of both species and a negative impact in photosynthetic-related parameters in P. radiata. We concluded that the rhizobiome of each pine species is distinct and higher abundance of bacterial taxa associated to disease protection was registered for the PPC-resistant species. Furthermore, differences in the rhizobiome are paralleled by a distinct content in phenolic compounds, which are also linked to plants’ resistance against PPC. This study unveils a species-specific rhizobiome and provides insights to exploit the rhizobiome for plant selection in nurseries and for rhizobiome-based plant-growth-promoting strategies, boosting environmentally friendly disease control strategies.

Tolerance of 2-benzoxazolinone and interactions with grass and pine hosts in a population of Fusarium circinatum

Fusarium circinatum, the causal agent of pitch canker in pines and a cryptic endophyte of grasses, was examined for heritable variation in tolerance of the grass defense compound 2-benzoxazolinone (BOA). A diverse population of F. circinatum progeny was assayed for growth rate on potato dextrose agar amended with BOA. Matings were conducted to allow for selection of progeny with lower and higher tolerance of BOA. The results confirmed heritable variation in BOA tolerance in F. circinatum. A subset of differentially tolerant progeny was used for inoculations of growth chamber grown Zea mays and greenhouse grown Pinus radiata. No differences were detected in the rate of infection or extent of colonization of Z. mays inoculated with F. circinatum progeny differing in tolerance of BOA. Pitch canker symptoms in inoculated P. radiata trees showed that high BOA tolerating isolates induced significantly longer lesion lengths than those induced by low BOA tolerating isolates. Results from this study were consistent with the proposition that F. circinatum evolved from grass colonizing ancestors, and that pathogenicity to pine is a relatively recent evolutionary innovation.

Monterey pine forest made a remarkable recovery from pitch canker

Monterey pine (Pinus radiata) is a species of limited distribution, with three native populations in California. In 1986, a disease known as pitch canker, caused by the fungus Fusarium circinatum, was identified as the cause of extensive mortality in planted Monterey pines in Santa Cruz County. Monitoring studies on the Monterey Peninsula documented rapid progression of the disease in the native forest during the 1990s, with most trees sustaining some level of infection. However, between 1999 and 2013, the severity of pitch canker stabilized, with many previously diseased trees then free of symptoms, and plots monitored between 2011 and 2015 documented a steady decline in the occurrence of new infections. Consequently, whereas pitch canker was once a conspicuous visual blight in the forest, by the end of the observation period, symptomatic trees had become a rarity. The arrested development of pitch canker is suggestive of a reduction in the frequency and duration of fog near the coast, which provides conditions necessary for the pathogen to establish infections.

Global Geographic Distribution and Host Range of Fusarium circinatum, the Causal Agent of Pine Pitch Canker

Fusarium circinatum, the causal agent of pine pitch canker (PPC), is currently one of the most important threats of Pinus spp. globally. This pathogen is known in many pine-growing regions, including natural and planted forests, and can affect all life stages of trees, from emerging seedlings to mature trees. Despite the importance of PPC, the global distribution of F. circinatum is poorly documented, and this problem is also true of the hosts within countries that are affected. The aim of this study was to review the global distribution of F. circinatum, with a particular focus on Europe. We considered (1) the current and historical pathogen records, both positive and negative, based on confirmed reports from Europe and globally; (2) the genetic diversity and population structure of the pathogen; (3) the current distribution of PPC in Europe, comparing published models of predicted disease distribution; and (4) host susceptibility by reviewing literature and generating a comprehensive list of known hosts for the fungus. These data were collated from 41 countries and used to compile a specially constructed geo-database. A review of 6297 observation records showed that F. circinatum and the symptoms it causes on conifers occurred in 14 countries, including four in Europe, and is absent in 28 countries. Field observations and experimental data from 138 host species revealed 106 susceptible host species including 85 Pinus species, 6 non-pine tree species and 15 grass and herb species. Our data confirm that susceptibility to F. circinatum varies between different host species, tree ages and environmental characteristics. Knowledge on the geographic distribution, host range and the relative susceptibility of different hosts is essential for disease management, mitigation and containment strategies. The findings reported in this review will support countries that are currently free of F. circinatum in implementing effective procedures and restrictions and prevent further spread of the pathogen.

Real-time loop-mediated isothermal amplification assay for rapid detection of Fusarium circinatum

Fusarium circinatum is the causal agent of pitch canker, a lethal disease of pine and other conifers. Since F. circinatum is a quarantine organism, its timely detection could efficiently prevent its introduction into new areas or facilitate spread management in already infected sites. In this study, we developed a sequence-specific probe loop-mediated isothermal amplification (LAMP) assay for F. circinatum using a field-deployable portable instrument. The assay was able to recognize the pathogen in host tissues in just 30 min, and the sensitivity of the assay made it possible to detect even small amounts of F. circinatum DNA (as low as 0.5 pg/μl). The high efficiency of this method suggests its use as a standard diagnostic tool during phytosanitary controls.

Compost Tea Reduces the Susceptibility of Pinus radiata to Fusarium circinatum in Nursery Production

Nutrition is one of the factors that most limits forestry plant growth; thus, current production in nurseries is based on conventional fertilization focused on enhancing vigor. However, an excessive intake of mineral nitrogen can cause morphological imbalances and the formation of more succulent tissues which, consequently, increase susceptibility to plant pathogens. Fusarium circinatum is the causal agent of pitch canker in plants of the Pinus genus, with Pinus radiata being the species most susceptible to this disease. This study compares the response of P. radiata seedlings to infection by F. circinatum as influenced by two fertilizers—conventional and aerated compost tea (ACT)—applied during the nursery phase. The potential of ACT against F. circinatum was first tested in vitro, where it was found to inhibit the pathogen’s mycelial growth and conidial germination. In the greenhouse, infected plants fertilized with ACT exhibited less severe internal and external symptoms of pitch canker and lower levels of pathogen colonization of both stems and needles than with conventional fertilizer. An analysis of the hormone content and defense-related gene expression shows greater salicylic acid production and phenylalanine ammonium-lyase and chalcone synthase expression in ACT-fertilized pine. All of the parameters assessed are consistent in showing that biofertilization with ACT reduces the susceptibility of pine seedlings to the disease compared with conventional fertilization.

Infection of the Native California Grass, Bromus carinatus, by Fusarium circinatum, the Cause of Pitch Canker in Pines

At Point Reyes National Seashore in California, Fusarium circinatum, the causal agent of pitch canker in pines, was isolated from Pinus muricata, the California native grass, Bromus carinatus, and the introduced grass, Holcus lanatus. All grass plants from which F. circinatum was isolated were symptomless. Pathogenicity of grass isolates was confirmed by inoculation of P. radiata trees, which developed symptoms similar to trees inoculated with a pine isolate of F. circinatum. Isolates from grasses were somatically compatible with isolates recovered from symptomatic pines. B. carinatus grown in a growth chamber was inoculated with a green fluorescent protein-expressing strain of F. circinatum. Segments of inoculated leaves were incubated in moist chambers; after 1 to 2 days, sporulating hyphae were observed growing from leaf tissue. Spores of F. circinatum removed from B. carinatus leaves were confirmed to be fluorescent when illuminated with ultraviolet light. These results raise the possibility that B. carinatus cryptically infected by F. circinatum may be a source of propagules capable of infecting pines.

Pine Pitch Canker (PPC): Pathways of Pathogen Spread and Preventive Measures

Fusarium circinatum (Nirenberg and O’ Donnell) is the causal agent of pine pitch canker (PPC) disease, one of the most devastating forest diseases worldwide. Long-distance spread occurs mainly through the movement of infected seeds whereas at regional level, the movement of seedlings, substrates, or containers may play an important role in fungal dispersal. Invasion of nurseries takes place via infected seeds and further spread can occur by planting contaminated seedlings, especially due to the possibility of infected plants remaining symptomless. Once established, F. circinatum spreads by rain, wind, and insects. The natural spread of the pathogen is limited due to the short dispersal distances of the spores and the fairly short flight distances of disseminating insects. In this review, we summarize the currently known dispersal pathways of the pathogen, discussing both natural and human-assisted processes. With the purpose of understanding how to best intervene in the disease’s development in nurseries and forests, we outline the epidemiology of the pathogen describing the key factors influencing its spread. Preventive measures to control the spread of F. circinatum locally and globally are described with special emphasis on the challenges in implementing them.

Preliminary assessment of pathogenicity of Fusarium circinatum on germlings of Pinus sylvestris in Ukraine

Fusarium circinatum is the causal agent of Pine Pitch Canker (PPC) of pines and established in the Iberian Peninsula in Europe only. However, it is hypothesized that its range could expand through the Europe in the near future. The disease is very harmful to forests and nurseries all around the world. Despite the aggressiveness of this fungus, no trials on the susceptibility of Ukrainian Scots pine provenances from Pinus sylvestris to F. circinatum have been performed. The first preliminary pathogenicity test was carried out in vitro and revealed the strong probability of high level of pine seedlings susceptibility to pitch canker.

Sampling and Detection Strategies for the Pine Pitch Canker (PPC) Disease Pathogen Fusarium circinatum in Europe

Fusarium circinatum Nirenberg & O’Donnel is listed among the species recommended for regulation as quarantine pests in Europe. Over 60 Pinus species are susceptible to the pathogen and it also causes disease on Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) and species in genera such as Picea and Larix. The European Food Safety Authority considers the probability of new introductions—via contaminated seeds, wood material, soil and growing substrates, natural means and human activities—into the EU very likely. Due to early detection, constant surveillance and control measures, F. circinatum outbreaks have officially been eradicated in Italy and France. However, the global spread of F. circinatum suggests that the pathogen will continue to be encountered in new environments in the future. Therefore, continuous surveillance of reproductive material, nurseries and plantations, prompt control measures and realistic contingency plans will be important in Europe and elsewhere to limit disease spread and the “bridgehead effect”, where new introductions of a tree pathogen become increasingly likely as new environments are invaded, must be considered. Therefore, survey programs already implemented to limit the spread in Europe and that could be helpful for other EU countries are summarized in this review. These surveys include not only countries where pitch canker is present, such as Portugal and Spain, but also several other EU countries where F. circinatum is not present. Sampling protocols for seeds, seedlings, twigs, branches, shoots, soil samples, spore traps and insects from different studies are collated and compiled in this review. Likewise, methodology for morphological and molecular identification is herein presented. These include conventional PCR with a target-specific region located in the intergenic spacer region, as well as several real-time PCR protocols, with different levels of specificity and sensitivity. Finally, the global situation and future perspectives are addressed.