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.     

Future outlook for Pinus patula in South Africa in the presence of the pitch canker fungus (Fusarium circinatum)

Approximately 50% of the area planted to softwood trees in South Africa has been established with Pinus patula, making it the most important pine species in the country. More effort has gone into developing this species for improved growth, tree form and wood properties than with any other species. This substantial investment has been threatened in the last 10 years by the pitch canker fungus, Fusarium circinatum. The fungus infects and contaminates nursery plants and, once transferred to the field, causes severe mortality of young trees in the first year after establishment. Although nurserymen have some control of the disease, it is recognised that the best long-term solution to mitigate damage because of F. circinatum infection is to identify tolerant species, clones and hybrids for deployment in plantations in the future. Research has shown that alternative species such as P. tecunumanii, P. maximinoi and P. elliottii are suitable for warm sites. Pine hybrids, particularly between P. patula and the high-elevation sources of P. tecunumanii, appear to be a suitable replacement on subtemperate and temperate sites. Although these alternative species and hybrids are more sensitive to subfreezing temperatures than P. patula, their planting range can be increased by including cold tolerance as a selection criterion. Future breeding efforts will most certainly focus on improving the tolerance of pure P. patula to F. circinatum, which can be achieved by identifying specific family crosses and tolerant clones. The commercial deployment of disease-tolerant control-pollinated P. patula and hybrid families will most likely be established as rooted cuttings, which requires more advanced propagation technology. In the long term, new seed orchards comprised of P. patula clones tolerant to F. circinatum could be used to produce seed for seedling production.     

Evidence for the occurrence of induced resistance to pitch canker,caused by Gibberella circinata (anamorph Fusarium circinatum), in populations of Pinus radiata

Pitch canker, caused by Gibberella circinata, was discovered in California in 1986. Although initially quite damaging to Monterey pines (Pinus radiata), the severity of pitch canker has moderated in areas where the disease was first observed and some trees appear to have recovered completely. The absence of symptoms on trees that were once severely affected implies they have become more resistant to the disease. Experimental work has shown that P. radiata can manifest systemic induced resistance (SIR) in response to infection by the pitch canker pathogen and observations of disease remission may indicate that SIR is operative under natural conditions as well. As a test of this hypothesis, the susceptibility of trees in remission was assessed by inoculating them with G. circinata and recording the extent of lesion development. In addition, randomly selected trees in areas that differed in residence time of pitch canker were inoculated to determine if trees with a longer period of exposure to the pathogen were more resistant to the disease. The results of these tests showed that 89% of trees observed to be in remission sustained very limited lesion development, consistent with resistance to pitch canker. Furthermore, trees in areas where pitch canker was well established tended to be more resistant than trees in areas where the disease was of more recent occurrence. In sum, these findings support the view that SIR occurs in P. radiata and is contributing to a moderation of the impact of pitch canker under natural conditions.     

Development of a quantitative real‐time PCR assay for the detection of Phytophthora austrocedrae,an emerging pathogen in Britain

A TaqMan real‐time PCR assay was developed for Phytophthora austrocedrae, an emerging pathogen causing severe damage to juniper in Britain. The primers amplified DNA of the target pathogen down to 1 pg of extracted DNA, in both the presence and absence of host DNA, but did not amplify any of the non‐target Phytophthora and fungal species tested. The assay provides a useful tool for screening juniper populations for the disease.     

Specific hybridization real‐time PCR probes for Phytophthora ramorum detection and diagnosis

Sudden Oak Death, caused by Phytophthora ramorum, poses a serious threat to native American oaks, and is also present in Europe where it has been isolated from numerous European ornamental plant nurseries. Its proven aggressiveness against plants in the Fagaceae and Ericaceae and the damage it has caused in North America have lead to it being assigned quarantine status. The timely and accurate detection of P. ramorum is a critical aid in the study of the epidemiology and biology of this pathogen. As a regulated organism, the availability of a sensitive and reliable assay is essential when attempting to achieve early detection of the pathogen. In this work, new specific hybridization probes for a real‐time PCR amplification method were found to be rapid, robust and labour‐saving, and proved suitable for routine use in a molecular diagnostic laboratory.     

Evaluation of Pinus radiata seed treatments to control Fusarium circinatum: effects on seed emergence and disease incidence

In this study, the effects of hot water (HWT), hydrogen peroxide and fungicides on the incidence of Fusarium circinatum on artificially inoculated Pinus radiata seeds were evaluated. Fifteen commercial fungicide formulations were screened in vitro for inhibitory activity on mycelial growth and conidial germination of F. circinatum. With half‐maximal effective concentration (EC₅₀) lower than 0.5 ppm, fluazinam, imazalil and tebuconazole were the most effective fungicides on mycelial growth, while captan, mancozeb or pyraclostrobin were the most effective (EC₅₀ < 0.3 ppm) on conidial germination. Based on the results obtained, imazalil, fluazinam, mancozeb and pyraclostrobin were selected for further testing. The effects of HWT, hydrogen peroxide and fungicide treatments on seed emergence and the incidence of F. circinatum were assessed. Seed treatments with fungicides prior to sowing were less effective and inconsistent in reducing the incidence of F. circinatum on seedlings. In contrast, hot water and hydrogen peroxide treatments significantly reduced F. circinatum contamination on P. radiata seeds with an overall disease incidence lower than 0.8% on seedlings. Furthermore, subsequent application of fungicides on seedlings did not improve the effectiveness of HWT. These results, therefore, suggest that hot water is a better alternative to hydrogen peroxide and fungicides as Pinus seed treatment against F. circinatum and could easily be implemented as standard in commercial nurseries to control the spread of the pitch canker disease.     

A new method to detect Lonsdalea quercina in infected plant tissues by real‐time PCR

Presymptomatic and accurate diagnoses of pathogens are essential for disease prediction and the timely application of bactericide. The bacterium Lonsdalea quercina (=Brenneria quercina) has been reported as the causal agent of drippy nut and bark canker disease on oak in California (US) and Europe. In recent years, it is also found on Populus × euramericana trees in Henan province of China. This bacterium causes longitudinal cankers of a few centimetres in size on the bark surface of the upper trunk. In this study, we developed two species‐specific PCR assays using primer pairs LqfF/LqfR and LqgF/LqgR for the rapid and accurate detection of the pathogenic bacteria in diseased plant tissues. The results show that the LqfF/LqfR primers amplified only a single PCR band of approximately 382 bp and the LqgF/LqgR primers yielded a PCR product of approximately 286 bp. The two primers were successfully adapted to real‐time PCR based on SYBR Green I used with the ABI 7500 system. The detection limit of the reaction was 0.1 pg genomic DNA per 20 μl PCR reaction volumes. The pathogen was mainly detected in the phloem of cankers as well as in the exudates of diseased trees, but was not found in the xylem or leaves. The size of pathogen in distribution was larger than the lesion. The results demonstrate that real‐time PCR assays can be used to detect the pathogen by extracting DNA directly from infected plant tissues. This method is a rapid, reliable method for the presymptomatic and accurate detection of L. quercina, providing a useful insight into epidemiological studies.     

Direct quantitative real‐time PCR assay for detection of the emerging pathogen Neonectria neomacrospora

The epidemic outbreak in northern Europe of Neonectria neomacrospora, the causal agent of dieback in Abies spp., led the European and Mediterranean Plant Protection Organization (EPPO) to include the pathogen on its alert list in 2017. Effective monitoring of this pathogen calls for a rapid and sensitive method of identification and quantification. A probe‐based real‐time PCR (qPCR) assay based on the β‐tubulin gene was developed for the detection and quantification of N. neomacrospora in infected wood samples, and directly for ascospores. This study presents the first published species–specific molecular detection assay for N. neomacrospora. The analytical specificity was validated on taxonomically closely related fungal species as well as on 18 fungal species associated with the host (Abies sp.). The analytical sensitivity was tested on naturally infected wood, on purified pathogen DNA in a matrix of host DNA and on N. neomacrospora ascospores for detection of airborne inoculum. The latter was tested both with a DNA extraction step prior to qPCR and without DNA extraction by direct qPCR on collected ascospores. The assay was specific to N. neomacrospora, with a sensitivity of 130 fg purified DNA, or 10 ascospores by direct qPCR. Omitting DNA extraction and amplifying directly on unpurified ascospores improved assay sensitivity significantly.     

Rapid diagnosis of pathogenic Phytophthora species in soil by real‐time PCR

Real‐time PCR assays based on the TaqMan system and using ITS sequences were developed for the identification of Phytophthora species, including P. cactorum, P. megasperma, P. plurivora, P. pseudosyringae and P. quercina, all of which are currently causing significant damage to roots of forest trees in both managed stands and natural ecosystems. Total genomic DNA was extracted from mycelia of aforementioned Phytophthora isolates. Species‐specific primers for P. cactorum, P. megasperma, P. plurivora, P. pseudosyringae and P. quercina were designed based on ITS sequences of rDNA. The amplification efficiency of target DNA varied from 93.1% (P. pseudosyringae) to 106.8% (P. quercina). The limit of the detection was calculated as 100 – 1,000 fg DNA, depending on the Phytophthora species. In mixed soil samples, all Phytophthora species were detected for Ct values shifted by 0.7 – 2.1 cycles. Based on these real‐time PCR assays we were able to identify the five Phytophthora species. These techniques will be of value in the identification of these pathogens, which may cause up to 80 – 90% fine root loss in oak stands.     

Methods for inoculum production and inoculation of Cistella japonica,the causal agent of resinous stem canker in Chamaecyparis obtusa

The ascomycete Cistella japonica was cultured on potato dextrose agar medium (PDA) for inoculation into Chamaecyparis obtusa, enabling the development of an inoculation method suitable for use in a breeding programme aimed at selecting for families of Ch. obtusa resistant to resinous stem canker. Using PDA to generate the inoculum resolved the problems encountered with the previously used mixed medium of rice bran and wheat bran, including unfavourable characteristics, uncertain growth of Ci. japonica mycelia, and a complex culturing operation. The inoculation test induced resin exudation similar to that observed in natural infections, and reproduced clonal differences with regard to damage severity.     

Rapid and accurate detection of Ceratocystis fagacearum from stained wood and soil by nested and real‐time PCR

A nested and real‐time PCR assay was developed for the rapid and accurate detection of Ceratocystis fagacearum, which is the causal agent of oak wilt in stained wood and soil. Based on the differences of the internal transcribed spacer (ITS) sequences of Ceratocystis spp., one pair of species‐specific primers, CF01/CF02, was designed. Whereas a 280‐bp product was amplified using the purified DNA from three isolates of C. fagacearum as the template, no PCR product was obtained from template of other 18 fungi. The detection sensitivity was 10 pg genomic DNA per 25‐μl PCR reaction volume. To increase detection sensitivity, a nested PCR was developed by using ITS1/ITS4 as the first‐round primers and CF01/CF02 in the second round, as it can detect 1 pg genomic DNA per 25‐μl PCR reaction volume. More importantly, CF01/CF02 primers were successfully adapted to real‐time PCR with a detection limit of 0.1 pg genomic DNA per 20‐μl PCR reaction volume. Using these two methods, we could rapidly and accurately detect the pathogen in artificially infected wood and soil.     

Molecular phylogeny and pathotyping of Fusarium solani: a causal agent of Dalbergia sissoo decline

Sissoo (Dalbergia sissoo), commonly known as shisham, is amongst the finest woods of South Asia, but ‘wilt’ disease has caused a rapid decline in this species. The cause of the disease remains uncertain. The aim of this study is to identify the causal agent of the disease and characterize isolates made from diseased trees, based on genomic data and variations in virulence. Samples of infected roots, stems and the ooze exuded from infected trees were obtained from plants showing symptoms in different geographical regions of India for the isolation of microorganisms. Isolates were used to inoculate healthy plants. Based on the morphological characteristics, genus‐ and species‐specific PCR, and in silico analysis of 5.8S rDNA‐ITS regions, of the 38 fungal isolates, 24 and 14 were identified as Fusarium solani and Fusarium sp., respectively. In a pathotyping study, eighteen F. solani isolates, isolated from roots and stem parts of symptomatic plants, induced typical wilt symptoms when inoculated through soil and roots on D. sissoo seedlings of 1–15 months in age. The population of F. solani was the highest in infected roots and the lowest in parts of stems, gradually decreasing with height, and was isolated constantly up to approximately 40% height of the seedling. F. solani isolates used in inoculations were successfully re‐isolated from the rhizosphere, infected roots and wilted stems, as confirmed using isolate‐specific DNA fingerprints. Molecular phylogenies based on rDNA‐ITS sequences showed that the 38 isolates fell into 2 groups. Group I comprised of F. solani isolates from D. sissoo and F. solani sequences in the NCBI GenBank database, whereas group II included Fusarium isolates other than F. solani. These results are helpful in developing integrated control measures for this highly variable pathogen and to establish a base for future population studies.     

A TaqMan real‐time PCR assay for the detection of Phytophthora ‘taxon Agathis’ in soil,pathogen of Kauri in New Zealand

Kauri Agathis australis, an iconic tree of New Zealand, is under threat from an introduced disease‐causing pathogen provisionally named Phytophthora ‘taxon Agathis’ (referred to as PTA). This soilborne, Pythiaceous species belongs to the Chromista and causes a collar rot resulting in yellowing of the foliage and thinning of the canopy, which eventually causes death of the infected tree. The management and containment of this pathogen requires rapid and reliable detection in the soil. The current method for soil detection utilizes a soil bioassay involving lupin baits and soil flooding in a process that takes between ten and twenty days. We describe a real‐time PCR assay based on TaqMan chemistry for the specific detection of PTA, which targets the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA. This TaqMan real‐time PCR assay could be used with DNA extracted directly from bulk soil samples to enable rapid quantification of PTA within soil. The detection limit was 2 fg of PTA DNA from pure culture, or 20 fg in the presence of DNA extracted from soil. The assay was validated using soil samples taken from a PTA‐infested site and soil spiked with a known concentration of oospores. We conclude that the TaqMan real‐time PCR assay offers a more time‐efficient method for detection of PTA in soil than existing methods.     

Detection method for Fusarium torreyae the canker pathogen of the critically endangered Florida torreya,Torreya taxifolia

Florida torreya (Torreya taxifolia Arn.) is an endangered conifer with a very limited range in the USA: two counties in Florida and one in Georgia, along the Apalachicola River. The species was once abundant in its small native range but suffered a major decline, ~99% loss, in the late 1950s to early 1960s that is thought to have been caused by a disease. Recently, a canker disease caused by Fusarium torreyae was identified as the primary cause of Florida torreya decline. Efforts to restore and preserve the species in situ and ex situ are hampered by lack of pathogen‐free planting stock, and there exists an interest in methods to verify pathogen presence in seeds and seedlings prior to collection and transport for planting. This paper presents a new species‐specific diagnostic method that enables detection of F. torreyae and may allow for conservation programmes to ensure germplasm is free of the pathogen prior to planting.     

Life cycle and in vitro sporulation dynamics of Corinectria constricta,the causal agent of Pinus radiata stem canker,in Chile

In 2008, a canker disease caused by the fungus Corinectria constricta was detected in southern Chile. The causal agent was previously identified as Neonectria fuckeliana (now Corinectria fuckeliana), which has been associated with stem cankers in Pinus radiata plantations in New Zealand since the 1990s. Many basic aspects of the life cycle of C. constricta remain unknown. The current study aimed to (a) document the periods during which C. constricta fruiting bodies are present in P. radiata plantations and associated factors; (b) determine the C. constricta life cycle in P. radiata plantations in southern Chile; and (c) evaluate, under in vitro conditions, the sporulation dynamics of ascospores. The first and second aims were carried out by evaluating affected plantations every 15 days, identifying asexual and sexual fungal structures, and recording the time periods when the structures were present. The third aim was achieved with in vitro tests in Petri dishes simulating humidity chambers. The life cycle was characterized by the presence of sporodochia from the Cylindrocarpon‐like (asexual form of C. constricta) morph during the autumn of 2012 (March–May). Subsequently, perithecia began to form on the sporodochia during April of 2012, taking approximately 3 months to mature (May–July), persisting for the rest of the year and providing inoculum to infect new trees. The development of perithecia in winter demonstrates that this is the most important period for dispersal and infection. In terms of sporulation dynamics, perithecia can release ascospores up to eight days following a wetting event; without this event, the spores are not released.     

Development of microsatellite markers for Thekopsora areolata,the causal agent of cherry spruce rust

Cherry spruce rust is a fungal disease of Norway spruce cones caused by Thekopsora areolata and responsible for significant losses in seed production in Sweden and Finland. Here, we report the first set of nine microsatellites, which will allow an effective genetic fingerprinting of T. areolata. The markers were isolated using the FIASCO method and were characterized using DNA from 49 single aecia sampled from spruce cones in three different seed orchards in Sweden. Eight of the nine markers were shown to be polymorphic among the aecia. The markers were unlinked and are therefore suitable for future population genetic studies.     

Investigation of the phenotypic and genetic properties of Brenneria nigrifluens strains,the causal agent of walnut bark canker in Kurdistan province,Iran

Phenotypic and genetic properties of Brenneria nigrifluens, the causal agent of walnut bark canker disease were studied using a combination of key biochemical tests, analysis of whole‐cell protein extracts and genetic fingerprints based on DNA primers corresponding to conserved motifs in bacterial repetitive (repetitive extragenic palindromic, enterobacterial repetitive intergenic consensus and BOX) elements and to the 16s rRNA gene. Phenotypic properties indicated that 11 strains showed a high degree of similarity and <5% of isolates showed different properties such as utilization of dulcitol and inositol. A majority of isolates produced rep‐PCR profiles similar (>80%) to a B. nigrifluens reference strain. This study showed that the majority of strains had 0–3 plasmids with estimated sizes ranging 9–32 Kb. Strains were clustered into two and three groups by whole‐cell protein analysis and rep‐PCR, respectively.     

A thirteen‐year study on the impact of a severe canker disease of Corymbia calophylla,a keystone tree in Mediterranean‐type forests

Worldwide, forests and woodlands have shown progressive declines in health as a result of global environmental changes in combination with local anthropogenic drivers. This study examined the incidence and progression of a canker disease of marri (Corymbia calophylla) caused by the endemic fungal pathogen Quambalaria coyrecup at three paired forest and anthropogenically disturbed sites in the southwest of Western Australia over 13 years. At the time of plot establishment in 2001, cankers were present on trees at all six sites with 22.7% of the assessed trees cankered. By 2014, cankers had led to the death of 6.7% of the trees, and an additional 10.0% of the trees developed cankers during this time. A further 2.3% of trees died due to causes other than canker, resulting in a final figure of 65.0% of trees remaining alive and free of cankers for the duration of the survey period. Canker incidence was significantly greater on trees present at anthropogenically disturbed sites (along roadsides and in paddocks) than forest trees (35.3% increasing to 50.7%, 10.0% increasing to 14.7%, respectively). Trunk diameter at breast height, tree height and crown ratings were not correlated with canker presence. This long‐term study provides evidence of the increasing severity of this canker pathogen and the impact it is having on the survival of marri.     

Testing of selected South African <Emphasis Type="Italic">Pinus</Emphasis> hybrids and families for tolerance to the pitch canker pathogen, <Emphasis Type="Italic">Fusarium circinatum</Emphasis>

Plantations of Pinus spp. constitute approximately 50% of the South African forestry industry. The first aim of this study was to develop a reliable inoculation technique to screen Pinus spp., for tolerance to infection by F. circinatum, which threatens pine forestry in South Africa. Inoculation of branches was compared with stem inoculations and we considered the number of branches or trees required to obtain statistically significant results. Furthermore, variation in the susceptibility of some Pinus families, clones and hybrids was considered. Results showed that branch inoculations were closely correlated with those from stem inoculations, and that it is important to consider branch and stem diameters when assessing susceptibility of trees. Subsequent trials using branch inoculations showed significant differences in F. circinatum tolerance amongst a range of pine species and hybrids of potential interest to forestry in South Africa. Significant differences in susceptibility were also found among clones of two P. radiata families. The most tolerant trees were P. elliottii × caribaea and P. patula × oocarpa hybrids, while the most susceptible species were P. patula, P. greggii and hybrids of these two. This is the first trial considering the susceptibility of Pinus hybrids, Pinus clones and some P. patula provenances, and the results indicate excellent potential for breeding for tolerance to pitch canker in South Africa. Application The accurate selection of disease tolerant planting stock for the South African forestry industry is crucially important for the continued sustainability of this important industry. The work described here provides valuable information on an artificial inoculation technique that will assist the industry in screening trees for tolerance to the pitch canker fungus, F. circinatum. It also provides some indication of the relative susceptibility of a number of Pinus spp., hybrids and families currently being evaluated in the country.