Biocontrol potential of Pseudomonas azotoformans,Serratia marcescens and Trichoderma virens against Fusarium wilt of Dalbergia sissoo

Wilt disease caused by Fusarium solani is a serious constraint to Dalbergia sissoo (shisham) plantations in northern India. In this study, the antagonistic potential of 40 bacterial isolates recovered from rhizophere soil of healthy shisham trees, and a well‐characterized Trichoderma species (Trichoderma virens) were tested for their possibility as biocontrol agents for F. solani. Two promising isolates (S1 and S15) were identified which inhibited pathogen growth, caused chitin degradation, produced siderophores and solubilized phosphate in vitro. Isolate S15 scored highest for hydrogen cyanide (HCN) production while isolate S1 was a non‐HCN producer. These two isolates were identified as Serratia marcescens (S1) and Pseudomonas azotoformans (S15) following sequence analysis of 16S rDNA. In dual culture assays, T. virens caused 80% inhibition of mycelial growth of the test fungus. The three selected antagonists when tested in planta in the glasshouse completely suppressed production of wilt symptoms on 12‐month‐old shisham plants. Further work is needed to ascertain the potential of these isolates to be used as biocontrol agents to manage shisham wilt under field conditions.     

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.     

First report of Lasiodiplodia theobromae and Fusarium proliferatum causing teak die back in Costa Rica

Teak plantation productivity and economic value have risen significantly since 2000; however, teak dieback syndrome in commercial plantations is becoming worse every year. We isolated, characterized and identified the causal agents of teak dieback, based on morphological characteristics in culture and DNA sequencing. The pathogenicity of Lasiodiplodia theobromae and Fusarium proliferatum (separate and together) was confirmed in inoculations of teak elite genotypes, fulfilling Koch's postulates. To our knowledge, this is the first report of L. theobromae and F. proliferatumcausing teak dieback syndrome in Costa Rica.     

First report of Lasiodiplodia theobromae and Pseudofusicoccum stromaticum causing dieback in Syzygium malaccense tree in Brazil

Syzygium malaccense is a woody species, known as Malay apple, which is grown as an ornamental or for fruit production in Brazil, mainly in the northern and north‐eastern regions. A high incidence of dieback has been observed in Malay apple in ornamental situations, including streets and gardens. Morphological identification, sequencing analysis of TEF+ITS gene regions and a pathogenicity test confirmed that fungi in the Botryosphaeriaceae cause the disease. This is the first report of Lasiodiplodia theobromae and Pseudofusicoccum stromaticum causing dieback on S. malaccense trees in Brazil.     

Fusarium species in declining wild apple forests on the northern slope of the Tian Shan Mountains in north‐western China

Wild apple forests in the Tian Shan Mountains in north‐western China have been adversely affected by an unknown disease in recent years. Symptoms attributed to this disease that affects wild apple trees include xylem browning and dieback which are suggestive of infection by Fusarium species. Therefore, the research team conducted the first survey for Fusarium in the afflicted wild apple forests. Twig samples with symptoms of xylem browning and dieback were collected in the Xinyuan, Gongliu, Yining and Huocheng Counties of Xinjiang Uighur Autonomous Region in China. Based on phylogenetic analyses and morphological observation, sixty strains of Fusarium accounted for 48% of the total number of fungi isolated from samples were subsequently classified into six species including twenty‐four F. avenaceum, seventeen F. solani, ten F. tricinctum, five F. proliferatum, two F. sporotrichioides and two unfamiliar Fusarium sp. 1. The five previously known species of Fusarium were then tested for pathogenicity to leaves and twigs in vitro. The results indicated that all of the species, except for F. tricinctum, can cause obvious lesions on the leaves of host plants and on the twigs of Fuji and wild apple. This is the first report of Fusarium species pathogenicity in Xinjiang wild apple forests, confirming a new host for these pathogens in this study.     

Fusarium equiseti is associated with the wilt and dieback of Aquilaria malaccensis in Northeast India

Aquilaria malaccensis, categorized by IUCN as globally vulnerable, is in high demand in the Middle East and Asian markets for its unique resinous agarwood. In August 2015, symptoms of dieback were observed on A. malaccensis trees planted in the Medicinal and Aromatic Plant Garden, Panbari, Golaghat of the Assam Forest Department. The entire crowns of 70 trees showed complete leaf loss and severe dieback. Rotting at the collar region and of roots was also observed. Isolation from the infected roots consistently yielded Fusarium equiseti identified following standard laboratory procedures and analysis of the internal transcribed spacer (ITS) sequence of the ribosomal DNA. Symptoms of wilt, dieback and root rot were observed on 5‐month‐old Aquilaria seedlings 25 days after inoculation with the isolated fungus. This paper is the first report of F. equiseti causing wilt and dieback of A. malaccensis.     

First report of Fusarium solani causing wilt of Melia dubia

Melia dubia, a multipurpose tree species, is gaining importance to meet the demand supply gap of timber, plywood and pulpwood . In June 2016, a serious outbreak of wilt disease was observed in M. dubia seedlings planted in the Central Nursery of Forest Research Institute (FRI), Dehradun, India. The disease led to the destruction of one hundred thousand (100,000) seedlings. Earlier in June 2012, serious wilting of M. dubia seedlings was observed in Haryana, India. The pathogen was identified as Fusarium solani following standard laboratory procedures and sequence analysis of the internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA). The pathogenicity of three isolates has been proved under greenhouse conditions. This is the first report of F. solani causing wilt of M. dubia.     

Differences in susceptibility to ash dieback in Czech provenances of Fraxinus excelsior

The impact of ash dieback caused by Hymenoscyphus fraxineus on 17 provenances of Fraxinus excelsior and one provenance of Fraxinus angustifolia was studied in an extensive field trial established in the Czech Republic prior to the H. fraxineus invasion in 1999. A difference in the level of resistance to ash dieback between the species was found: F. angustifolia was significantly less affected by the disease than F. excelsior. Moreover, particular provenances of F. excelsior showed important differences in the level of resistance to H. fraxineus. A relationship between the impact of ash dieback and altitude was also discovered – the provenances from altitudes above 600 m a.s.l. were less affected by the pathogen than were the provenances from lower areas. No difference in the impact of the disease among provenances of F. excelsior from different ecotopes (ravine, calcareous ravine and alluvial) was found. Substantial among‐tree variability in resistance to H. fraxineus was observed throughout the trial – promising genotypes (with crown defoliation up to 5%) were identified in all 18 tested provenances. In regard to this finding, it appears that the main source of resistance to the pathogen is probably at the individual genotype level in the trial. A secondary but massive attack by Hylesinus fraxini was identified in the trees that had been greatly damaged by ash dieback, and the beetle caused their health to deteriorate significantly. A significant negative effect of the presence of collar necroses caused by H. fraxineus and browse damage was also identified.     

Pathogenicity of ophiostomatoid fungi on Picea abies in Slovenia

Pathogenic fungi can survive and develop in living plants, often causing diseases in the host. Some theories speculate that pathogenic ophiostomatoid fungi provide benefits to its vectors – bark beetles – by overcoming the tree's defence mechanisms. This study reports the results of an experiment in south‐eastern Europe in which mature and seedling Norway spruce trees were artificially inoculated with various ophiostomatoid fungi. The aim of the experiment was to determine the relative virulence of ophiostomatoid fungi by assessing the ability of the fungi to stimulate host tree defence mechanisms through inoculation experiments. Experiments were performed by inoculation of Picea abies in seedling and mature trees. The following fungi were used in low‐density and seedling inoculations: Ophiostoma ainoae, O. brunneo‐ciliatum, Grosmannia cucullata and an unidentified Leptographium sp., O. bicolor, O. fuscum, O. piceae, G. penicillata and G. piceiperda. Endoconidiophora polonica was used in mass and seedling inoculations. Various characteristics such as host vitality, blue stain, lesion and resin outflow were measured before and after the trees were felled. E. polonica caused blue stain, induced large lesions and killed some of the mature trees and seedlings, confirming earlier reports that it is a strong wound pathogen. Only E. polonica, Leptographium sp. and O. ainoae caused blue stains in the sapwood of inoculated seedlings. In low‐density inoculations, G. piceiperda induced intense necrosis and had higher values for all the characteristics monitored. Some of the other ophiostomatoid fungi showed a moderate level of pathogenicity. Fungi with the capacity to stimulate a host defence mechanism could play a role in the establishment of bark beetle populations.     

Hymenoscyphus fraxineus can directly infect intact current‐year shoots of Fraxinus excelsior and artificially exposed leaf scars

Hymenoscyphus fraxineus, the causal agent of ash dieback, was inoculated onto intact, unwounded current‐year shoots and leaf scars of 4‐year‐old, potted Fraxinus excelsior seedlings. Pieces of ash wood colonized by the fungus were used as inoculum. Three of 25 (12%) of the inoculated intact shoots and nine of 25 (36%) of the inoculated leaf scars were infected by H. fraxineus and developed typical symptoms of ash dieback, including necrotic lesions on the shoot surface and wood discoloration as well as shoot and leaf wilting distal to the inoculation site. No symptoms occurred on control seedlings, which had been inoculated in the same way but with sterile wood pieces. Visible necrotic lesions on shoots and wood discoloration were statistically significantly longer in proximal than in distal direction from the inoculation site, a pattern which resembles symptoms after natural infection. The ash dieback pathogen was re‐isolated from nine of 12 (75%) of the symptomatic seedlings. These results provide indirect supportive evidence that the fungus infects shoots via leaves and shows that it is able, under experimental conditions using a massive mycelial inoculum, to directly infect intact, unwounded current‐year shoots of its main host in Europe.     

Resistance of Dalbergia sissoo to Fusarium solani f.sp. dalbergiae

Large‐scale dieback of Dalbergia sissoo trees in India and neighbouring countries is causing concern to the state forest departments and farmers as trees of all age group are dying. Fusarium solani f.sp. dalbergiae, isolated from the roots of dying trees and from the oozing pitch on stems, was tested for pathogenicity. Seeds were collected from 107 mature healthy trees, from 21 heavily infected localities in the country. As many seed failed to germinate, resistance testing was carried out on a limited number of provenances. Seedlings were tested for disease resistance by soil drenching and root‐dip methods using spore suspensions of F. solani f.sp. dalbergiae. On the basis of seedling survival, provenances were classified into four susceptibility classes; seedlings from Amritsar (Punjab) showed maximum survival and were considered very resistant, whereas Dehra (Kangra), Himachal Pradesh, were the most susceptible. F. solani f.sp. dalbergiae from Nihal Gate (Haldwani), Uttarakhand, was the most virulent isolate amongst the three tested.     

Fungal colonization patterns in necrotic rootstocks and stem bases of dieback‐affected Fraxinus excelsior L.

European ash (Fraxinus excelsior) trees currently face the major threat of ash dieback caused by an invasive fungus, Hymenoscyphus fraxineus. Collar rots in F. excelsior have been increasingly associated with infections by this pathogen. However, the aetiology of the collar rots is still unclear and remains heavily debated. In contrast to most studies of this kind, entire rootstocks of four diseased ash trees were dug out to examine necrotic tissues in these rootstocks and stem bases in detail and to sample necrotic wood for fungal isolation. With the aid of morphological and molecular identification techniques, five to twelve fungal taxa were detected per tree. Members of the Nectriaceae family and Botryosphaeria stevensii, the causal agent of stem and branch cankers on many tree species, were frequently isolated from outer xylem. In contrast, H. fraxineus was the dominating species in interior wood layers. Microsatellite genotyping of 77 H. fraxineus isolates helped to identify up to six different genotypes per tree. The role of H. fraxineus and other isolated fungi in the aetiology of ash collar rots are discussed.     

First report of Cryptosphaeria pullmanensis as causal agent of Cryptosphaeria canker of Populus nigra in Iran

A diatrypaceous fungus was isolated consistently from cankers on Populus nigra trees showing dieback symptoms in Kohgiluyeh Boyer‐Ahmad, Zanjan and Esfahan provinces in Iran. Morphological characteristics and phylogenetic analyses of the ITS region of the rDNA identified the taxon as Cryptosphaeria pullmanensis. Pathogenicity tests conducted in potted 3‐month‐old cuttings of P. nigra confirmed Koch's postulates and revealed that C. pullmanensis caused canker on this host. This is the first report of C. pullmanensis causing a canker disease on P. nigra in Iran.     

Identification and characterization of fungi associated with internal wood lesions and decline disease of willow and poplar trees in Iran

During the study of fungal trunk pathogens associated with urban trees decline in Shiraz (Iran), a serious decline of willow and poplar trees was observed. Therefore, an investigation was conducted on these trees in some areas of this city during spring and summer 2012 and 2013, to determine the main fungal trunk pathogens associated with these ornamental plants. Plant materials were collected from trees exhibiting disease symptoms such as yellowing, shoot canker, shoot dieback, defoliation and internal wood necrosis and decayed wood. Fungal isolations were made from discoloured or decayed wood tissue onto 2% malt extract agar (MEA) amended with streptomycin sulphate. Nine species, Fomes fomentarius, Diplodia seriata, Lasiodiplodia theobromae, Dothiorella sarmentorum, Neoscytalidium hyalinum, Diatrype whitmanensis, Phaeoacremonium rubrigenum, P. aleophilum and P. parasiticum, were identified based on morphology and DNA sequence comparisons. Pathogenicity tests were performed on detached shoots of willow and poplar trees under greenhouse conditions. Lasiodiplodia theobromae caused the longest lesions on willow. On poplar shoots, the longest lesions were caused by P. parasiticum. Diplodia seriata produced the smallest lesions on both woody hosts. First reports from willow wood include P. parasiticum, P. rubrigenum, D. whitmanensis, L. theobromae, D. seriata and N. hyalinum, while new reports from poplar wood include P. parasiticum and Do. sarmentorum. Based on our knowledge, this is also the first report of D. whitmanensis in Iran.     

Pathogenicity of Malaysian Phytophthora palmivora on cocoa,durian, rubber and oil palm determines the threat of bud rot disease

The epidemic of bud rot disease affecting oil palm in Colombia is primarily caused by Phytophthora palmivora. The pathogen has a cosmopolitan presence that includes Southeast Asia, but to date, bud rot has not been reported in this region. This study provides an overview of the potential risk of Malaysian P. palmivora isolates cross‐infecting other host species, including cocoa, durian, rubber and Malaysian oil palm planting materials (Dura × Pisifera, D × P). On cocoa pods, the durian isolate PP7 caused dark brown necrotic lesions. Detached leaf bioassays showed that P. palmivora isolates PP3 and PP7 infected different hosts, except rubber foliage without wounding. Inoculation tests on cocoa, durian and rubber seedlings caused brown necrotic lesions when stems were wounded, with 10% mortality in cocoa and durian at 17 days post‐inoculation (dpi). However, no further infection was observed, and lesions closed within 14–28 dpi on the non‐wounded seedlings. Pathogenicity tests of oil palm seedlings inoculated with isolates PP3 and PP7 indicated that Malaysian P. palmivora isolates were not pathogenic to oil palms based on localized infection observed only through wounding. Overall, the work demonstrated that Malaysian P. palmivora isolates were able to cross‐infect multiple hosts but did not show severe infections on oil palms.     

Pathogenicity to alder of Phytophthora species from riparian ecosystems in western Oregon

Described as one of the most destructive pathogens of agricultural crops and forest trees, Phytophthora is a genus of microorganisms containing over 100 known species. Phytophthora alni has caused collar and root disease in alders throughout Europe, and a subspecies has recently been isolated in North America. Reports of canopy dieback in red alder, Alnus rubra, prompted a survey of their overall health and to determine whether P. alni was present in western Oregon riparian ecosystems. Over 1100 Phytophthora isolates were recovered, representing 20 species and 2 taxa. Phytophthora‐type cankers were observed in many trees, and their incidence was positively correlated with canopy dieback. High levels of mortality for red alder were not observed, which suggests these Phytophthora species are not aggressive pathogens. To test this hypothesis, three stem wound inoculations and one root dip were conducted on red alder seedlings using 13 Phytophthora species recovered from the riparian survey. Ten of the 13 Phytophthora species produced significant lesions in at least one pathogenicity test. Phytophthora siskiyouensis produced the largest lesions on red alder from the two stem wound inoculation tests conducted under summer conditions, while P. taxon Pgchlamydo caused the largest lesions during the winter stem wound inoculation test. Phytophthora gonapodyides, P. taxon Pgchlamydo and P. siskiyouensis have previously been found associated with necrotic alder roots and bole cankers in the field, and with the pathogenicity results reported here, we have established these species as causes of Phytophthora root disease and Phytophthora bole canker of alder in Oregon. While none of the Phytophthora species were especially aggressive towards red alder in the pathogenicity tests, they did cause localized disease symptoms. By weakening the root systems or boles of alders, the Phytophthoras could be leaving alders more susceptible to other insects and pathogens.     

Hymenoscyphus pseudoalbidus and Hymenoscyphus albidus: viridiol concentration and virulence do not correlate

Hymenoscyphus pseudoalbidus is the causal agent of ash dieback, a disease that is presently endangering Fraxinus spp. throughout most of Europe. The phytotoxin, viridiol, was previously isolated from culture extracts of H. pseudoalbidus and found to be toxic to leaves of F. excelsior. Thus, we were interested in learning to what extent viridiol is responsible for pathogenicity of H. pseudoalbidus and investigated this using twelve isolates of H. pseudoalbidus. We also included five isolates of the closely related avirulent species, Hymenoscyphus albidus, in our studies. Some, but not all, isolates of H. pseudoalbidus and H. albidus produced measurable quantities of viridiol in culture. Three tests were used to determine to what extent viridiol concentration correlates with virulence: culture extracts were tested for activity in leaf segment tests and for inhibition of germination of seedlings of Fraxinus excelsior; virulence of the isolates was tested following infection of axenically cultured ash seedlings. Activity of the culture extracts varied, as did virulence of the isolates following inoculation into seedlings. No correlations were found between viridiol concentration and activities of culture extracts in leaf segment tests or in the germination test, nor between viridiol concentration and disease symptoms when inoculated into seedlings. However, activities of culture extracts in leaf segment and in the germination test correlated, as did the results of each of these tests with virulence in the infection experiment. Apparently, as yet unidentified factors other than the concentration of viridiol play important roles in the virulence of H. pseudoalbidus.     

Occurrence and pathogenicity of fungi in necrotic and non-symptomatic shoots of declining common ash (<Emphasis Type="Italic">Fraxinus excelsior</Emphasis>) in Sweden

Currently, massive dieback of Fraxinus excelsior is observed in countries of eastern, northern and central Europe, and the reasons for it are unclear. The aims of the present work were (a) to study fungal communities in declining F. excelsior crowns; (b) to clarify role of fungi in the decline. Shoots from symptomatic crowns were collected in four localities in central Sweden, and distributed into the following categories: (a) visually healthy; (b) initial necroses; (c) advanced necroses; (c) dead tops. The most frequently isolated fungi were Gibberella avenacea, Alternaria alternata, Epicoccum nigrum, Botryosphaeria stevensii, Valsa sp., Lewia sp., Aureobasidium pullulans and Phomopsis sp., and these taxa were consistently found in shoots of all four symptomatic categories. Forty-eight taxa of other fungi were isolated, and fungal diversity was not exhausted by the sampling effort. The same taxa of fungi were dominant in F. excelsior shoots of different symptomatic categories, and moderate to high similarity of fungal communities was observed in shoots despite the symptoms. Forty-four isolates from 24 fungal taxa were used for artificial inoculations of 277 1-year-old F. excelsior seedlings in bare root nursery. After 2 years, only four fungi caused symptomatic necroses of bark and cambium: A. alternata, E. nigrum, Chalara fraxinea and Phomopsis sp. The most pathogenic was C. fraxinea, inducing symptoms on 50% of inoculated trees, while three other fungi caused necroses on 3–17% of inoculated trees. Infection biology of C. fraxinea and environmental factors determining susceptibility of F. excelsior to decline deserve future investigations.     

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.