Identification of Colletotrichum species associated with anthracnose of Spondias spp. in Brazil

Plants of the genus Spondias are commonly cultivated in northern and north‐eastern Brazil for fruit production. Severe leaf anthracnose, caused by Colletotrichum spp., is frequently observed in several species of Spondias. The objectives of this study were to identify and characterize Colletotrichum species associated with anthracnose in species of Spondias by using the concept of morphological and phylogenetic analyses. Leaves with symptoms of anthracnose were collected from orchards in the state of Piauí, Brazil. Morphological identification; sequencing analysis of ACT, ITS and GS gene regions; and a pathogenicity test confirmed three Colletotrichum spp. (C. dianesei, C. siamense and C. brevisporum) were able to cause the disease. This is the first record of these species of Colletotrichum causing anthracnose in Spondias spp. in Brazil.     

Assessment of Colletotrichum gloeosporioides as a biological control agent for management of hemlock dwarf mistletoe (Arceuthobium tsugense)

The ascomycete Colletotrichum gloeosporioides was tested for biological control of Arceuthobium tsugense on western hemlock (Tsuga heterophylla) in British Columbia (BC), Canada. A field trial was conducted near Nanaimo, BC, using C. gloeosporioides isolate PFC 2415 in three treatments applied in late August 2002. The treatments consisted of C. gloeosporioides formulated with ‘Stabileze’ (incorporation of fungi in a water‐absorbent starch matrix with oil and sucrose, then granulating the matrix with hydrated silica) which were sprayed on (1) intact mistletoe swellings, (2) swellings on which all mistletoe shoots had been cut at 0.5 cm from the base of the shoot and (3) C. gloeosporioides formulated in a sucrose and gelatin preparation sprayed on intact mistletoe swellings. The ultimate goal of this research was to substantially reduce the ability of the treated dwarf mistletoe plants to produce seed inoculum. The ‘Stabileze’ and sucrose–gelatin treatments formulated with C. gloeosporioides reduced the current berry crop by 36.8 and 40.5%, respectively (p < 0.05). While the results for shoots appeared promising, heavy background infection and/or secondary infection, especially on controls, limited the ability to detect clear treatment effects. Careful culturing from various live and dead host tissues showed that C. gloeosporioides was unable to invade and kill the mistletoe endophytic system within the living xylem and phloem of the host.     

Blight of conifer seedlings caused by Colletotrichum gloeosporioides

In a growth chamber experiment, 70-day-old seedlings of 10 conifer species were inoculated with Colletotrichum gloeosporioides conidia to determine the host range of the fungus. Based on the percentage of seedlings affected and the disease severity on individual seedlings, the order of most to least susceptibility was: western hemlock (WH), mountain hemlock, western larch, Sitka spruce, Engelmann spruce, Douglas-fir (coastal form, then interior form), white spruce and ponderosa pine; lodgepole pine and western red cedar remained unaffected. Inoculation of WH needles showed that within 24 h C. gloeosporioides conidia germinate and appressoria (penetration structures) form. A growth chamber study demonstrated that the pathogen can infect WH at needle wetness periods as short as 15 min; number of needles affected was higher at 0.5 h, but did not increase further even when wetness was extended up to 8 h. The results are discussed in relation to blight management of greenhouse-grown conifer seedlings.Portion of a Bachelor of Science (Honors) thesis, Department of Biology, University of Victoria, Victoria, British Columbia.     

Biochemical responses associated with induced resistance to Colletotrichum acutatum in Pinus radiata seedlings treated with methyl jasmonate and Trichoderma spp.

The effect of Trichoderma (T. atrobrunneumFCC320 and T. atrovirideLU633) and/or methyl jasmonate (MJ) on resistance to terminal crook (Colletotrichum acutatum) and on seedling biochemistry was investigated in radiata pine (Pinus radiata) seedlings. Seedlings were germinated and grown in Trichoderma‐amended or non‐amended media for 3 months and then sprayed with 2.25 mM MJ 1 week before inoculation with C. acutatum. The incidence and severity of terminal crook in the seedlings treated with MJ and Trichoderma+MJ were lower than in Trichoderma‐treated and Trichoderma‐untreated seedlings. The MJ‐induced resistance response was concomitant with an increase in the concentrations of the monoterpenes α‐pinene, β‐pinene, β‐phellandrene, camphene and myrcene in needles, and also α‐pinene, β‐pinene and camphene in stems. The concentrations of α‐pinene, β‐pinene and camphene were elevated from at least 1 week until 4 weeks after MJ application, compared with those in non‐MJ counterparts. Trichoderma alone did not affect monoterpenes, but the concentrations of α‐pinene, β‐pinene and camphene were greater in needles of Trichoderma+MJ than in MJ‐treated seedlings after 28 days. Total phenolic concentration in needles and peroxidase activity in stems were twofold greater in MJ‐treated seedlings than in non‐MJ seedlings over the same period. None of the treatments affected the activity of peroxidase in needles. It is proposed that the accumulation of monoterpene and phenolics and the induction of peroxidases contribute, in part, to MJ‐induced resistance to terminal crook in radiata pine seedlings.     

Black canker and leaf spot of Salix in New Zealand caused by Glomerella miyabeana (Colletotrichum gloeosporioides)

Two new diseases have been observed attacking willows in New Zealand. In the first instance black stem cankers formed on S. matsudana × S. alba × S. alba cultivars, eventually killing them. In the second instance foliage of S. gooddingii and S. amygdaloides was attacked and premature defoliation occurred. Morphological studies and host inoculations established that Glomerella miyabeana (Colletotrichum gloeosporioides anamorph) was the causal agent of both diseases. Foliage of cultivars exhibiting stem cankering was not attacked nor were stems of cultivars with foliar lesions. Electron microscope studies showed that conidia of Colletotrichum gloeosporioides formed phialidically within acervuli and comdial dimensions varied significantly with host.     

An in vitro method for screening Colletotrichum gloeosporioides as a biological control agent for western hemlock dwarf mistletoe

An in vitro system using detached western hemlock branches infected with dwarf mistletoe was developed to screen the virulence of five isolates of Colletotrichum gloeosporioides, a hyperparasite of dwarf mistletoe shoots and berries. Detached branches infected with dwarf mistletoe were placed in nutrient‐saturated rock‐wool blocks and mistletoe shoots were inoculated with a conidial suspension of C. gloeosporioides. One month after inoculation, lesions on mistletoe stems and berries were well developed. Infection levels for individual isolates varied from 40% to 60% of shoots and 60% to 80% of berries. Significant differences were found between the isolates and control (p = 0.02 and 0.001, respectively) while no differences were noted between the isolates for both the shoots and berries. Parallel inoculation of mistletoe shoots detached from hemlock branches on moist filter paper and in rock‐wool blocks failed because these shoots deteriorated rapidly, fragmenting into segments within a week. This in vitro test may provide an alternative method for rapid screening of potentially virulent C. gloeosporioides isolates.     

Infection of Arceuthobium americanum by Colletotrichum gloeosporioides and its potential for inundative biological control

Inundative biological control of Arceuthobium americanum occurring on Pinus contorta var. latifolia with the fungus Colletotrichum gloeosporioides was investigated. Isolates of C. gloeosporioides were collected throughout British Columbia, Canada, and one isolate was selected for assessment based on its growth and sporulation in culture. The fungus was formulated using the ‘Stabileze’ method and inoculated onto A. americanum under field conditions. It became established on some replicates and there was a higher incidence of C. gloeosporioides on treated replicates than controls. In some replicates, the treatment reduced fruit production, leading to a decrease in the reproductive capacity of the dwarf mistletoe plant; however, the efficacy was highly variable and not significant.     

‘Pink rot’: infection of Castanea sativa fruits by Colletotrichum acutatum

Pink‐coloured tissues were observed in rotting chestnut nuts collected from soil in different orchards in Italy (Emilia Romagna, Tuscany and Trentino). Morphological and molecular analysis confirmed the presence of Colletotrichum acutatum (J.H. Simmons) in affected fruits, and inoculation tests corroborated the ability of the pathogen to colonize nuts.     

Enhancing resistance in Pinus radiata seedlings to terminal crook (Colletotrichum acutatum) using methyl jasmonate and ultraviolet‐C radiation

Colletotrichum acutatum is a fungal pathogen that causes terminal crook disease in radiata pine (Pinus radiata) seedlings in New Zealand forest nurseries. Symptoms of infection include malformation or death of the growing tip and a stiffening and thickening of the stem. Although the disease can be managed effectively using fungicides, the New Zealand forest industry is interested in alternative control options such as induced resistance. The aim of this study was to evaluate spray application of chitosan (1.4 g/l) or 2.25 mm methyl jasmonate (MeJA) and irradiation with UV‐C (2.16 kJ/m²) for their potential to induce resistance to terminal crook. The treatments were applied to 4‐month‐old seedlings at 1 week before pathogen inoculation. By the end of the experimental period (42 days after inoculation), there was 80% disease incidence in the controls, with 48% of seedlings exhibiting severe terminal crook symptoms. The most effective treatment (p < 0.05) was MeJA with 16% disease incidence and none with severe symptoms. UV‐C also significantly (p < 0.05) reduced infection with 52% incidence and 20% of seedlings exhibiting severe symptoms. Chitosan did not reduce disease incidence (72%) compared with the control (80%) but did significantly reduce (p < 0.05) disease severity with 28% exhibiting severe symptoms. MeJA was the only treatment that significantly (p < 0.05) reduced the detrimental effects of infection on seedling apical growth and stem diameter. To our knowledge, this is the first report demonstrating the potential for MeJA and UV‐C to control terminal crook in radiata pine.     

Colletotrichum theobromicola causing anthracnose on butia fruits in Brazil

Native fruits of Brazil have received more attention recently due to their organoleptic properties and as sources of secondary metabolites that improve human health. Amongst the native fruit species are Butia spp palms, the fruits of which are consumed raw or processed into juices, jams, liquor and ice cream. In this work, anthracnose was found on fruits from Butia odorata plants. Fruits heavily affected by this condition are considered inappropriate for consumption of raw or use in processed foods. The fungus was identified using morphological and molecular methods as Colletotrichum gloeosporioides, phylogenetically close to Colletotrichum theobromicola. To the best of our knowledge, this is the first report of C. theobromicola causing anthracnose in Butia species.     

Genetic transformation,electrophoretic karyotyping and isolation of insertional mutants in the tree pathogenic fungus Neonectria galligena

The ascomycete tree pathogen Neonectria galligena was transformed using several plasmid vectors containing the bacterial hygromycin phosphotransferase gene (hph) conferring resistance to hygromycin B. Protoplasts were produced by digesting germinating conidia in a Novozym 234 solution osmotically stabilized with NaCl. The protoplasts regenerated at ca. 70% on complete medium supplemented with either sucrose or NaCl. Up to 115 hygromycin B‐resistant transformants per microgram of transforming DNA per 10⁸ protoplasts were obtained. Southern analysis of genomic DNA from the transformants, separated by either standard agarose gel electrophoresis or by pulsed field gel electrophoresis, confirmed that hygromycin B resistance resulted from integration of the vector. Transformants expressing the green fluorescent protein were also obtained. A total of 209 transformants were then screened for aggressiveness and mycelial radial growth. Four mutants were identified including one mutant with reduced aggressiveness but normal growth in vitro, two mutants with reduced growth in vitro but normal aggressiveness, and one mutant in which both aggressiveness and in vitro growth were reduced.     

Agrobacterium tumefaciens‐mediated transformation for targeted disruption and over expression of genes in the poplar pathogen Sphaerulina musiva

Sphaerulina musiva causes both leaf spots and cankers on poplar. Leaf spots can lead to defoliation and cankers on branches and primary stems can lead to stem breakage and tree mortality. The recent availability of both the S. musiva and Populus trichocarpa genomes offers a great opportunity to study host–pathogen interactions. To better understand the factors involved in S. musiva pathology, we present a strategy for the transformation of this species using Agrobacterium tumefaciens. Binary plasmids were generated with hygromycin B phosphotransferase (hph) flanked by upstream and downstream sequences of polyketide synthase‐like (PKS‐L1) gene to generate targeted gene disruptants by homologous recombination. Plasmids were also constructed for constitutive expression reporter genes eGFP and mCherry to help with histological characterization of the pathogen during infection. Gene knockouts were identified by PCR and confirmed by sequencing and Southern blotting. No significant differences were observed in melanin production between PKS‐L1 disruptants and wild type isolates. Colonies expressing reporter genes were identified by fluorescent stereomicroscopy. This method is a promising tool for the characterization of pathogen genes through reverse and forward genetics and for introducing markers for histopathological study.     

Biochemical response and induced resistance against anthracnose (Colletotrichum camelliae) of camellia (Camellia pitardii) by chitosan oligosaccharide application

This study demonstrated that disease resistance against anthracnose (caused by Colletotrichum camelliae) in camellia (Camellia pitardii) could be induced by chitosan oligosaccharide (COS) applied to leaves at 50 µg/ml. The induced resistance lasted for 10–15 days, with the optimum induced effect against anthracnose occurring by the third day. Following treatment with C. camelliae alone, COS alone and C. camelliae and COS combined, camellia plants showed different responses, which included the activity of H₂O₂, the defence‐related enzymes, such as polyphenol oxidase, peroxidase, catalase, phenylalanine ammonialyase, and concentration of soluble proteins. Plants that received COS treatments consistently had higher levels of biochemical activity than plants not treated with COS. Assessment of anthracnose severity on infected plants, as evidenced by reduced lesions on leaves, indicated that COS‐induced camellia plants had significantly lower disease ratings. To our knowledge, we are first to report the inducement by COS on camellia against anthracnose.     

A major facilitator superfamily transporter in Colletotrichum fructicola (CfMfs1) is required for sugar transport,appressorial turgor pressure,conidiation and pathogenicity

The major facilitator superfamily (MFS) is one of the largest membrane‐protein families. To investigate the role of MFS proteins in the fungal plant anthracnose pathogen Colletotrichum fructicola, the CfMFS1 gene was deleted. This resulted in reduced mycelial growth, conidial yield and decreased virulence on tea oil camellia leaves. In addition, ?Cfmfs1 showed increased sensitivity to osmotic stress and to a cell‐wall stressor. Further analysis revealed that CfMfs1 is required for conidial penetration and appressorial turgor pressure, both important for fungal pathogen invasion. Confocal fluorescence microscopy showed that CfMfs1 is localized to membranes of both hyphae and conidia, suggesting that it may be a membrane transporter. Our study provides evidence that CfMfs1 has a role in conidiation, sugar transport, stress response, conidial penetration, appressorial turgor pressure and virulence against tea oil camellia.     

qPCR quantification of Ophiognomonia clavigignenti‐juglandacearum from infected butternut trees under different release treatments

The use of a molecular assay for quantifying conidia of Ophiognomonia clavigignenti‐juglandacearum, the fungal pathogen responsible of butternut canker, was investigated. Purified DNA from conidia collected on glass fibre filters of a passive rain collectors was quantified using a TaqMan real‐time quantitative polymerase chain reaction (qPCR) assay. The qPCR assay could specifically discriminate the target species from all other North American known species of Ophiognomonia, and it was sensitive enough to repeatedly detect one conidium. A linear relationship between numbers of conidia and qPCR C_t values was determined, and used to assess the sporulation of the pathogen under trees that were released to promote their vigour. In total, 977 samples of field‐captured conidia from 49 trees, at two locations, and from two successive growing seasons were analysed. No significant difference of sporulation was observed under control and release treatments. However, our results demonstrated that qPCR assay was reliable for detecting and quantifying O. clavigignenti‐juglandacearum from environmental samples, which will be useful to assess further control methods for this disease.     

The use of the green fluorescent protein as a biomarker for sapstain fungi

To understand wood colonization by sapstain fungi and their potential biocontrol agents, it is necessary to differentiate these organisms directly on their natural substrates. In the present study the feasibility of transforming with the green fluorescent protein (GFP), the sapstain fungus Ophiostoma piceae and a potential biocontrol agent Cartapip^®, an Ophiostoma piliferum albino strain was assessed. Transformants of the two fungal species were screened by polymerase chain reaction and Southern blot analyses. The GFP was expressed in spores, synnemata and mycelia of the transformants grown in artificial media or wood. The growth, pigmentation and wood colonization of the transformants were similar to that of the non‐transformants, suggesting that the presence of the gfp gene had no negative effect on the biology of the transformants. Using fluorescence and confocal microscopy, the GFP‐expressing fungi were easily differentiated from the wild‐type strains and other fungal species in wood, even 4 months after inoculation. The results show that the use of the GFP system is feasible to monitor Ophiostoma fungi in wood.     

Identification and pathogenicity of Alternaria alternata causing leaf spot and blight disease of Ailanthus excelsa in India

Alternaria leaf spot of Ailanthus excelsa is generally considered as minor disease in India. Recently, severe disease outbreaks were recorded in the nursery of the Forest Research Institute, Dehradun, progeny trial at Jhumpa, Haryana, and in a nearby farm field. Leaf symptoms included small circular, brown, necrotic spots with a chlorotic halo. With severe infections, leaf spots coalesced and resulted in leaf blight. A small‐spored Alternaria with concatenated conidia was isolated consistently from the leaf samples with spot symptoms. Sequence analyses of the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) and the translation elongation factor 1‐alpha (tef‐1α) gene region of two fungal isolates confirmed the species as A. alternata. In detached leaf assays, both isolates produced symptoms similar to those observed on the nursery/field‐grown plants. To validate the detached leaf assay result, pathogenicity was also demonstrated on whole plants in a glasshouse. Koch's postulates were fulfilled by re‐isolating A. alternata from the inoculated leaves. This work is the first to confirm that A. alternata is associated with leaf spot and blight disease of A. excelsa in India.     

Rapid detection of Fusarium circinatum propagules on trapped pine beetles

Pitch canker is a destructive disease of pine caused by the fungus Fusarium circinatum. This taxon is listed as a quarantine fungus for several regional plant protection organizations throughout the world. Whereas long‐distance spread of the disease is made possible through the trade of infected pine seeds, local spread is caused by aerial dispersion or insect transportation of the fungal conidia. Developing a reliable and efficient tool to detect of F. circinatum in insects would be very useful to monitor the local spread of the pathogen. This tool would also provide the means to assess the range of insect species that could serve as potential vector of the fungus. A DNA extraction protocol was optimized and combined with a real‐time PCR test to detect F. circinatum on pine beetles. Using artificially contaminated Ips sexdentatus, it was shown that the test was able to detect down to 10 F. circinatum conidia per individual, and 20 conidia per batch of 10 insects, which is below the lowest inoculum load occurring in nature. With this technique, several batches of up to 10 insects may be analysed simultaneously, with a timescale for analysis reduced to <5 h and without the need for expertise in Fusarium taxonomy. This tool may be useful to monitor potential spread of the pathogen across regions. Using this method, to date, despite F. circinatum foci occurred in Northern Spanish regions across the border in France, the pathogen was not found on I. sexdentatus.     

Characterization and pathogenicity of six Cytospora strains causing stem canker of wild apple in the Tianshan Forest,China

Cytospora species are capable of causing destructive cankers of stems belonging to a wide range of woody plant species. In severe cases, cankers may lead to dieback of twigs and branches. Little is known about the Cytospora species causing canker disease of wild apple (Malus sieversii) trees in the Wild Fruit Forest Reserve in Tianshan Forest, Xinjiang Uygur Autonomous Region, China. In this study, six Cytospora isolates belonging to two species were isolated from cankerous lesions of wild apple twigs. Based on multi‐locus phylogenetic analysis using three DNA markers (ITS, tef1‐α and tub2) and morphological characterization, these isolates were identified as Cytospora mali and Cytospora parasitica. Temperature trials (15, 20, 25 and 30°C) showed that the optimal growth temperature for six isolates was 25°C. At a variety of temperatures, C. mali isolates tended to grow faster than isolates of C. parasitica, with the C. mali isolate, EGI1 performing better than others with regard to growth rate. Morphological observations showed that these species exhibited a single locule without conceptacles, and the conidia length was 3–5 μm. In vitro inoculation trials of twigs and leaves of M. sieversii seedlings revealed that the C. mali isolates were highly virulent phytopathogenic fungi, whereas the C. parasitica isolates were less virulent. The isolate EGI1 was the most virulent isolate among the six isolates. This paper presents the first report of pathogenic Cytospora spp. of the M. sieversii Tianshan Wild Fruit Forest Reserve of Yili, Xinjiang in China. It will aid in the understanding of how apple tree cankers are induced and provide disease management guidelines for M. sieversii forest conservation.     

Colletotrichum siamense: A novel leaf pathogen of Sterculia nobilis Smith detected in China

Sterculia nobilis is an important tropical woody plant with high ornamental and economic value. At present, there are few reports of diseases on this plant. In August 2018, an unknown leaf spot disease was observed on S. nobilis in Nanning, China, affecting the inner leaves of the canopy and causing considerable defoliation. The pathogen was isolated, and pathogenicity tests carried out on detached leaves to verify Koch's postulates. Based on morphological observations and polygenic analyses, the pathogen causing leaf spots on S. nobilis was identified as Colletotrichum siamense.