Phylogeny, Morphology, Distribution, and Pathogenicity of Seven Calonectria Species from Leaf-Blighted Eucalyptus in HaiNan Island, China.

Leaf blight caused by Calonectria species constrains Eucalyptus trees in China. Calonectria leaf disease on Eucalyptus in China was first reported in HaiNan Island in 1985. No systematic investigation of Calonectria species associated with diseased Eucalyptus in HaiNan has been performed. To understand the species diversity, distribution, and pathogenicity of these Calonectria, 400 Calonectria isolates were obtained from 278 diseased Eucalyptus planted in 17 sites in five regions. All 400 isolates were identified by DNA sequences of translation elongation factor 1-alpha, ß-tubulin, calmodulin, and histone H3 gene regions and on morphology. Seven species, C. acaciicola (198 isolates), C. pseudoreteaudii (161 isolates), C. reteaudii (29 isolates), C. hawksworthii (6 isolates), C. hongkongensis (4 isolates), C. auriculiformis (1 isolate), and C. chinensis (1 isolate), were identified. This is the first report of C. acaciicola in China. C. acaciicola, C. pseudoreteaudii, and C. reteaudii belong to the C. reteaudii species complex and accounted for 97% of all isolates. The three species overlapped in vesicle shape, macroconidia size, and macroconidia septa number. Region significantly influenced C. acaciicola and C. pseudoreteaudii distribution. Representative isolates of C. acaciicola, C. pseudoreteaudii, C. reteaudii, and C. hawksworthii producing abundant macroconidia were used in conidial suspension inoculation on Eucalyptus seedlings; all were highly pathogenic to the two tested genotypes. The tolerances of two Eucalyptus genotypes were significantly different. This first systematic investigation of Calonectria species associated with Eucalyptus leaf blight in HaiNan will aid selection of disease-resistant genotypes for managing Eucalyptus leaf blight caused by Calonectria species in China.

Multi-Year Field Plantings Evaluating Boxwood Cultivars for Susceptibility to the Blight Pathogens (Calonectria spp.) in Northern Germany.

Two multiyear field trials were conducted to evaluate boxwood cultivars for their susceptibility to the blight pathogens Calonectria pseudonaviculata and C. henricotiae in northern Germany. Fifteen cultivars were included in the first trial from 2007 to 2012, and 46 cultivars were included in the second trial from 2014 to 2017. Both trials were done in a naturally infested field that was supplemented with infected plant tissue added to the soil before planting. Each cultivar had three replicate hedge sections with 10 plants per section, and they were assessed annually for blight severity expressed as proportion of leaves blighted and fallen. Blight severity varied significantly among years (P < 0.0001) and cultivars (P < 0.05) within each trial. In the first trial, mean severity ranged from 0.03 to 0.11 for the most resistant cultivars and 0.35 to 0.96 for the most susceptible ones. Similarly, in the second trial, mean severity ranged from 0.06 to 0.27 and 0.71 to 0.97 for the most resistant and susceptible cultivars, respectively. 'Suffruticosa' was consistently the most susceptible cultivar, followed by 'Marianne', 'Myosotidifolia', 'Raket', and 'Morris Midget'. 'Herrenhausen' was the most resistant cultivar, followed by B. microphylla var. japonica, B. microphylla var. koreana, 'Green Mound', 'Faulkner', and 'Winter Beauty'. This study provides field data showing the performance of boxwood cultivars under different levels of disease pressure in an area where C. henricotiae was dominant. This knowledge will help boxwood growers and gardeners to choose less susceptible cultivars and help plant breeders to select for disease resistance.

Virulence-Associated Genes of Calonectria ilicola, Responsible for Cylindrocladium Black Rot.

The Cylindrocladium black rot caused by Calonectria ilicicola is a destructive disease affecting a broad range of crops. Herein, we study virulence-associated genes of C. ilicicolaCi14017 isolated from diseased peanut roots (Arachis hypogaea L.). Ci14017 was identified via phylogenetic analysis of the internal transcribed spacer region and standard Koch's postulate testing. Virulence-associated genes were based on genome analyses and comparative analysis of transcriptome and proteome profiles of sensitive and resistant peanut cultivars. Ci14017 identified as C. ilicicola has a 66 Mb chromosome with 18,366 predicted protein-coding genes. Overall, 46 virulence-associated genes with enhanced expression levels in the sensitive cultivars were identified. Sequence analysis indicated that the 46 gene products included two merops proteins, eight carbohydrate-active enzymes, seven cytochrome P450 enzymes, eight lipases, and 20 proteins with multi-conserved enzyme domains. The results indicate a complex infection mechanism employed by Ci14017 for causing Cylindrocladium black rot in peanuts.

Evaluation of Fungicide Soil Drench Treatments to Manage Black Root Rot Disease of Avocado.

Four common fungicidal products were evaluated for their effect on symptoms caused by two nectriaceous black root rot fungi, Calonectria ilicicola and Dactylonectria macrodidyma, when applied as pot drenches to avocado (Persea americana) seedlings in the greenhouse. Applications of fludioxonil, thiophanate-methyl + etridiazole, prochloraz, and prochloraz MnCl at 2 and 4 weeks after inoculation with C. ilicicola significantly reduced root necrosis and improved root and aboveground plant biomass compared with water-treated controls. Fludioxonil reduced necrosis by 60% and had a significantly lower frequency of reisolation of C. ilicicola than the other three fungicide treatments. D. macrodidyma inoculation caused less severe symptoms in seedlings than C. ilicicola despite the longer duration of the trial. Pot drenches with fludioxonil, thiophanate-methyl + etridiazole, and prochloraz MnCl, but not prochloraz alone, significantly reduced root necrosis caused by D. macrodidyma. Prochloraz MnCl was the only fungicide treatment to increase root and plant biomass compared with water-treated controls. Both fludioxonil and prochloraz MnCl reduced the frequency of reisolation of D. macrodidyma from necrotic roots by about 50% compared with the other fungicides or water controls. The results indicated that drenches with these fungicides may suppress existing low to moderate black root rot infection, allowing new root growth and improved establishment in the orchard. Fungicide drenching must not replace best-practice disease management strategies in nurseries but may be a useful tool in crisis situations.

Calonectria in the age of genes and genomes: Towards understanding an important but relatively unknown group of pathogens.

The genus Calonectria includes many aggressive plant pathogens causing diseases on various agricultural crops as well as forestry and ornamental tree species. Some species have been accidentally introduced into new environments via international trade of putatively asymptomatic plant germplasm or contaminated soil, resulting in significant economic losses. This review provides an overview of the taxonomy, population biology, and pathology of Calonectria species, specifically emerging from contemporary studies that have relied on DNA-based technologies. The growing importance of genomics in future research is highlighted. A life cycle is proposed for Calonectria species, aimed at improving our ability to manage diseases caused by these pathogens.

Assessment of Selected Commercially Available Seed Treatments on Suppressing the Effects of Red Crown Rot on Soybeans Under a Controlled Environment.

Calonectria ilicicola is a soilborne fungus responsible for red crown rot (RCR) in soybeans. Recently, this disease has been detected in new areas within the United States and Asia, where it has been reported to cause significant yield losses. To date, no data on the efficacy of fungicide seed treatments for suppressing RCR in soybeans are available. We screened three commercially available soybean seed treatments (Acceleron STANDARD; Acceleron STANDARD + IleVO; Cruiser MAXX + Saltro) for impacts on germination, seedling growth, and disease caused by C. ilicicola under controlled laboratory conditions. The Cruiser MAXX + Saltro treatment improved final plant dry mass to levels similar to noninoculated controls. Both Acceleron STANDARD + IleVO and Cruiser MAXX + Saltro reduced percent root rot. Acceleron STANDARD alone did not impact root rot, indicating that the IleVO component was efficacious on C. ilicicola. These data indicate that IleVO and Cruiser MAXX + Saltro have potential for reducing early season impacts of RCR in soybeans. Future work assessing these seed treatments in field-grown soybeans is needed to better understand the role of seed treatments as components of an integrated management program for RCR.

Soil-borne Calonectria (Hypocreales, Nectriaceae) associated with Eucalyptus plantations in Colombia.

Eucalyptus spp. are widely planted in Colombia as an important component of a growing paper and pulp industry. Leaf and shoot blight caused by Calonectria spp. was one of the first disease problems to emerge in these plantations. A survey of Eucalyptus plantations in four forestry regions of Colombia during 2016 resulted in a large number of Calonectria isolates from soil samples collected in the understories of trees having symptoms of Calonectria leaf and shoot blight. The aim of this study was to identify and resolve the phylogenetic relationships for these isolates using DNA sequence comparisons of six gene regions as well as morphological characters. From a collection of 107 isolates, seven Calonectria species residing in three species complexes were identified. Two of these represented undescribed species, namely C.exiguispora sp. nov. and C.guahibo sp. nov. Calonectriaparvispora and C.spathulata were the most commonly isolated species, each of which accounted for approximately 30% of the isolates. The results suggest that Colombia has a wide diversity of Calonectria spp. and that these could challenge Eucalyptus plantation forestry in the future.

Two new species of Calonectria (Hypocreales, Nectriaceae) causing Eucalyptus leaf blight in Brazil.

In recent decades, commercial Eucalyptus plantations have expanded toward the warm and humid regions of northern and northeastern Brazil, where Calonectria leaf blight (CLB) has become the primary fungal leaf disease of this crop. CLB can be caused by different Calonectria species, and previous studies have indicated that Calonectria might have high species diversity in Brazil. During a disease survey conducted in three commercial plantations of Eucalyptus in northeastern Brazil, diseased leaves from Eucalyptus trees with typical symptoms of CLB were collected, and Calonectria fungi were isolated. Based on phylogenetic analyses of six gene regions (act, cmdA, his3, rpb2, tef1, and tub2) and morphological characteristics, two new species of Calonectria were identified. Five isolates were named as C.paragominensis sp. nov. and four were named as C.imperata sp. nov. The pathogenicity to Eucalyptus of both species was confirmed by fulfilling the Koch's postulates.

Comparative analysis of extracellular proteomes reveals putative effectors of the boxwood blight pathogens, Calonectria henricotiae and C. pseudonaviculata.

Calonectria henricotiae (Che) and C. pseudonaviculata (Cps) are destructive fungal pathogens causing boxwood blight, a persistent threat to horticultural production, landscape industries, established gardens, and native ecosystems. Although extracellular proteins including effectors produced by fungal pathogens are known to play a fundamental role in pathogenesis, the composition of Che and Cps extracellular proteins has not been examined. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and bioinformatics prediction tools, 630 extracellular proteins and 251 cell membrane proteins of Che and Cps were identified in the classical secretion pathway in the present study. In the non-classical secretion pathway, 79 extracellular proteins were identified. The cohort of proteins belonged to 364 OrthoMCL clusters, with the majority (62%) present in both species, and a subset unique to Che (19%) and Cps (20%). These extracellular proteins were predicted to play important roles in cell structure, regulation, metabolism, and pathogenesis. A total of 124 proteins were identified as putative effectors. Many of them are orthologs of proteins with documented roles in suppressing host defense and facilitating infection processes in other pathosystems, such as SnodProt1-like proteins in the OrthoMCL cluster OG5_152723 and PhiA-like cell wall proteins in the cluster OG5_155754. This exploratory study provides a repository of secreted proteins and putative effectors that can provide insights into the virulence mechanisms of the boxwood blight pathogens.

Species Diversity, Mating Strategy and Pathogenicity of Calonectria Species from Diseased Leaves and Soils in the Eucalyptus Plantation in Southern China.

Many Calonectria species are causal agents of diseases on several forestry, agricultural and horticultural crops. Calonectria leaf blight is one of the most important diseases associated with Eucalyptus plantations and nurseries in Asia and South America. Recently, symptoms of leaf rot and leaf blight caused by Calonectria species were observed in a one-year-old Eucalyptus experimental plantation in GuangXi Province, southern China. To better understand the species diversity, mating strategy and pathogenicity of Calonectria species isolated from diseased tissues and soils, diseased leaves and soils under the trees from ten Eucalyptus urophylla hybrid genotypes were collected. Three hundred and sixty-eight Calonectria isolates were obtained from diseased Eucalyptus leaves and soils under these trees, and 245 representative isolates were selected based on the sampling substrates and Eucalyptus genotypes and identified by DNA sequence analyses based on the translation elongation factor 1-alpha (tef1), ß-tubulin (tub2), calmodulin (cmdA) and histone H3 (his3) gene regions, as well as a combination of morphological characteristics. These isolates were identified as Calonectria hongkongensis (50.2%), C. pseudoreteaudii (47.4%), C. aconidialis (1.6%), C. reteaudii (0.4%) and C. auriculiformis (0.4%). This is the first report of C. reteaudii and C. auriculiformis occurrence in China. Calonectria pseudoreteaudii was isolated from both Eucalyptus diseased leaves and soils; the other four species were only obtained from soils. MAT1-1-1 and MAT1-2-1 gene amplification and mating type assignment results showed that C. pseudoreteaudii is heterothallic and an asexual cycle represents the primary reproductive mode, C. reteaudii and C. auriculiformis are likely to be heterothallic and C. hongkongensis and C. aconidialis are homothallic. Based on the genetic diversity comparisons for C. pseudoreteaudii isolates from diseased leaves and soils, we hypothesize that C. pseudoreteaudii in soils was spread from diseased leaves. Both the mycelia plug and conidia suspension inoculations indicated that all five Calonectria species were pathogenic to the two Eucalyptus genotypes tested and the tolerance of the two genotypes differed. It is necessary to understand the ecological niche and epidemiological characteristics of these Calonectria species and to select disease resistant Eucalyptus genotypes in southern China in the future.

Global Genetic Diversity and Mating Type Distribution of Calonectria pauciramosa: An Important Wide-Host-Range Plant Pathogen.

The fungal pathogen, Calonectria pauciramosa, has caused serious diseases of many important plants worldwide. Understanding the genetic diversity and mating type distribution of this pathogen provides an essential step toward the development of disease control measures. In this study, we designed 15 polymorphic microsatellite markers by using genome sequences of two Ca. pauciramosa isolates having opposite mating type and from different countries. These markers were used to determine the genetic diversity of 145 isolates representing 13 different hosts (12 plant hosts residing in 12 genera, and soil) from 10 countries. In addition, mating type genes were amplified to investigate the reproduction mode of the pathogens in these populations by using mating type primers designed for Calonectria spp. Results revealed that a single dominant genotype, isolated from 11 plant genera residing in eight families, was present in seven countries across five continents. Only mating type MAT1-1 or MAT1-2 was amplified in each of the isolates, confirming that Ca. pauciramosa is heterothallic. Both mating types were detected in isolates from Eucalyptus in South Africa and Uruguay. The MAT1-2 phenotype was widely distributed in isolates from 12 different hosts (11 plant hosts and soil) collected in 10 countries. Overall, the results suggest that there has been substantial global movement of Ca. pauciramosa and that this has shaped its current population structure.

Indications of Susceptibility to Calonectria pseudonaviculata in Some Common Groundcovers and Boxwood Companion Plants.

Knowing the host range of a pathogen is critical to developing and implementing effective disease management programs. Calonectria pseudonaviculata (Cps) is known to attack a number of species, varieties, and cultivars in the genus Buxus as well as three Pachysandra species (Pachysandra terminalis, Pachysandra procumbens, and Pachysandra axillaris) and several Sarcococca species, all in the Buxaceae family. The objective of this study was to evaluate non-Buxaceae groundcovers and companion plants commonly associated with boxwood plantings for their susceptibility to Cps. Twenty-seven plant species belonging to 21 families were exposed to different levels of inoculum: 50 to 300 conidia per drop for detached leaf assays and 30,000 to 120,000 conidia per 1 ml for whole-plant assays. Inoculated plants were incubated in humid environments for at least 48 h to facilitate infection. Cps infection and sporulation were observed on 12 plant species: Alchemilla mollis, Arctostaphylos uva-ursi, Brunnera macrophylla, Epimedium × youngianum, Galium odoratum, Geranium sanguineum, Phlox subulata, Tiarella cordifolia, Callirhoe involucrata, Iberis sempervirens, Mazus reptans, and Vinca minor. These results suggest that there may be more hosts of Cps commonly grown in nurseries and landscapes. If corroborated by observations of natural infection, these findings have implications for the Boxwood Blight Cleanliness Program instituted by the National Plant Board and for planning disease mitigation at production and in the landscape.

A Potent Burkholderia Endophyte against Boxwood Blight Caused by Calonectria pseudonaviculata.

Calonectria pseudonaviculata (Cps) poses an increasing threat to boxwood, a major nursery crop and iconic landscape plant worldwide. Here, we report on a potent biocontrol agent that produces small sage green (SSG) colonies on potato dextrose agar. SSG is a bacterial strain recovered from Justin Brouwers boxwood leaves with unusual response to Cps inoculation. Water-soaked symptoms developed on leaves 2 days after inoculation then disappeared a few days later. This endophyte affected several major steps of the boxwood blight disease cycle. SSG at 107 cfu/mL lysed all conidia in mixed broth culture. SSG at 108 cfu/mL reduced blight incidence by >98% when applied one day before or 3 h after boxwood were inoculated with Cps. Its control efficacy decreased with decreasing bacterial concentration to 103 cfu/mL and increasing lead time up to 20 days. When applied on diseased leaf litter under boxwood plants, SSG reduced Cps sporulation and consequently mitigated blight incidence by 90%. SSG was identified as a new member of the Burkholderia cepacia complex with distinct characters from known clinical strains. With these protective, curative, and sanitizing properties, this Burkholderia endophyte offers great promise for sustainable blight management at production and in the landscape.

Reconsideration of species boundaries and proposed DNA barcodes for Calonectria.

Calonectria represents a genus of phytopathogenic ascomycetous fungi with a worldwide distribution. In recent years, there has been an increase in the number of taxonomic studies on these fungi. Currently, there are 169 described species of Calonectria based on comparisons of DNA sequence data, combined with morphological characteristics. However, for some of these species, the sequence data utilised at the time of their description were relatively limited. This has justified an urgent need to reconsider the species boundaries for Calonectria based on robust genus-wide phylogenetic analyses. In this study, we utilised 240 available isolates including the ex-types of 128 Calonectria species, and re-sequenced eight gene regions (act, cmdA, his3, ITS, LSU, rpb2, tef1 and tub2) for them. Sequences for 44 Calonectria species, for which cultures could not be obtained, were downloaded from GenBank. DNA sequence data of all the 169 Calonectria species were then used to determine their phylogenetic relationships. As a consequence, 51 species were reduced to synonymy, two new species were identified, and the name Ca. lauri was validated. This resulted in the acceptance of 120 clearly defined Calonectria spp. The overall data revealed that the genus includes 11 species complexes, distributed across the Prolate and Sphaero-Naviculate Groups known to divide Calonectria. The results also made it possible to develop a robust set of DNA barcodes for Calonectria spp. To accomplish this goal, we evaluated the outcomes of each of the eight candidate DNA barcodes for the genus, as well as for each of the 11 species complexes. No single gene region provided a clear identity for all Calonectria species. Sequences of the tef1 and tub2 genes were the most reliable markers; those for the cmdA, his3, rpb2 and act gene regions also provided a relatively effective resolution for Calonectria spp., while the ITS and LSU failed to produce useful barcodes for species discrimination. At the species complex level, results showed that the most informative barcodes were inconsistent, but that a combination of six candidate barcodes (tef1, tub2, cmdA, his3, rpb2 and act) provided stable and reliable resolution for all 11 species complexes. A six-gene combined phylogeny resolved all 120 Calonectria species, and revealed that tef1, tub2, cmdA, his3, rpb2 and act gene regions are effective DNA barcodes for Calonectria.

Mating genes in Calonectria and evidence for a heterothallic ancestral state.

The genus Calonectria includes many important plant pathogens with a wide global distribution. In order to better understand the reproductive biology of these fungi, we characterised the structure of the mating type locus and flanking genes using the genome sequences for seven Calonectria species. Primers to amplify the mating type genes in other species were also developed. PCR amplification of the mating type genes and multi-gene phylogenetic analyses were used to investigate the mating strategies and evolution of mating type in a collection of 70 Calonectria species residing in 10 Calonectria species complexes. Results showed that the organisation of the MAT locus and flanking genes is conserved. In heterothallic species, a novel MAT gene, MAT1-2-12 was identified in the MAT1-2 idiomorph; the MAT1-1 idiomorph, in most cases, contained the MAT1-1-3 gene. Neither MAT1-1-3 nor MAT1-2-12 was found in homothallic Calonectria (Ca.) hongkongensis, Ca. lateralis, Ca. pseudoturangicola and Ca. turangicola. Four different homothallic MAT locus gene arrangements were observed. Ancestral state reconstruction analysis provided evidence that the homothallic state was basal in Calonectria and this evolved from a heterothallic ancestor.

Synthesis and bioactivity of indoleacetic acid-carbendazim and its effects on Cylindrocladium parasiticum.

Indoleacetic acid (IAA)-carbendazim was synthesized to assess whether this conjugate could retain the fungicidal activity of carbendazim and gain root-inducing properties upon the addition of an indoleacetic acid group. An indoor virulence test demonstrated that the conjugate retained the fungicidal activity of carbendazim towards Cylindrocladium parasiticum. The conjugate was detected in roots after soaking Ricinus communis L. leaves into a solution of the IAA-carbendazim, which confirmed its phloem mobility. The activities of the cellulase, polygalacturonase and xylanase produced by Cylindrocladium parasiticum treated with different concentrations of the conjugate were determined, and the peak activities appeared at 72 h or 96 h. More importantly, the conjugate showed the ability to promote root growth. These results revealed that indoleacetic acid-carbendazim may be useful in preventing Cylindrocladium parasiticum and other diseases.

Host responses and impact on the boxwood blight pathogen, Calonectria pseudonaviculata.

MAIN CONCLUSION: Boxwood leaves are more susceptible to Calonectria pseudonaviculata (Cps) and better suited for Cps reproduction than those of pachysandra and sweet box. Passages through a non-boxwood host may alter Cps ability to sporulate. Calonectria pseudonaviculata (Cps) infects boxwood and its two common companion plants-pachysandra and sweet box. This study investigated how boxwood, pachysandra, and sweet box respond to Cps isolates of different host origin. Detached leaves were inoculated with nine isolates, three from each host, and evaluated for colonization, infection rate, lesion size, and production of conidia and microsclerotia. Cps colonized boxwood leaf tissue within 12 h of inoculation, and 60 h ahead of pachysandra and sweet box. Cps also produced significantly larger lesions and more conidia on boxwood than on pachysandra and sweet box. Isolates originating from different host plants did not differ in all the components evaluated except for conidia production. Isolates from boxwood and sweet box produced significantly more conidia than those from pachysandra. Overall, boxwood leaves are more susceptible to the disease and are better suited for Cps reproduction than those of pachysandra and sweet box. Passages through a non-boxwood host may alter Cps ability to sporulate. These results advance the understanding of Cps biology and affirm the importance of taking pachysandra and sweet box into consideration in disease management planning.

Calonectria species isolated from Eucalyptus plantations and nurseries in South China.

Diseases caused by species of Calonectria (Ca.) represent a serious threat to the growth and sustainability of Eucalyptus plantations in China. Symptoms caused by these fungi mainly include leaf blight on trees in plantations and rotting of stems and leaves in nurseries. Extensive surveys have recently been conducted where Calonectria species were collected in Eucalyptus plantations and nurseries in the FuJian, GuangDong, GuangXi, and YunNan Provinces of South China. Additional isolates were baited from soil samples in the Hong Kong Region. The aim of this study was to identify the 115 Calonectria isolates obtained using comparisons of DNA sequence data for the ß-tubulin (tub2), calmodulin (cmdA), histone H3 (his3) and partial translation elongation factor-1α (tef1) gene regions as well as their morphological features. Seven known species were identified, including Calonectria arbusta, Ca. asiatica, Ca. chinensis, Ca. eucalypti, Ca. hongkongensis, Ca. mossambicensis and Ca. pentaseptata. In addition, six novel taxa were collected and are described here as Ca. aciculata, Ca. honghensis, Ca. lantauensis, Ca. pseudoturangicola, Ca. pseudoyunnanensis, and Ca. yunnanensis spp. nov. Overall, the results reflect a high diversity of Calonectria species in China.

Development of Nested PCR, Multiplex PCR, and Loop-Mediated Isothermal Amplification Assays for Rapid Detection of Cylindrocladium scoparium on Eucalyptus.

Eucalyptus dieback disease, caused by Cylindrocladium scoparium, has occurred in last few years in large Eucalyptus planting areas in China and other countries. Rapid, simple, and reliable diagnostic techniques are desired for the early detection of Eucalyptus dieback of C. scoparium prior to formulation of efficient control plan. For this purpose, three PCR-based methods of nested PCR, multiplex PCR, loop-mediated isothermal amplification (LAMP) were developed for detection of C. scoparium based on factor 1-alpha (tef1) and beta-tubulin gene in this study. All of the three methods showed highly specific to C. scoparium. The sensitivities of the nested PCR and LAMP were much higher than the multiplex PCR. The sensitivity of multiplex PCR was also higher than regular PCR. C. scoparium could be detected within 60 min from infected Eucalyptus plants by LAMP, while at least 2 h was needed by the rest two methods. Using different Eucalyptus tissues as samples for C. scoparium detection, all of the three PCR-based methods showed much better detection results than regular PCR. Base on the results from this study, we concluded that any of the three PCR-based methods could be used as diagnostic technology for the development of efficient strategies of Eucalyptus dieback disease control. Particularly, LAMP was the most practical method in field application because of its one-step and rapid reaction, simple operation, single-tube utilization, and simple visualization of amplification products.