In vitro screening of biocontrol and biodegradation potential of selected hymenomycetes against Ganoderma boninense and infected oil palm waste

The antagonistic and enzymatic activities of non‐pathogenic hymenomycetes naturally occurring on oil palm (Elaeis guineensis) trunks were assessed to determine their potential as biocontrol agents against G. boninense. Twenty‐five Basidiomycota were isolated on a Ganoderma‐selective medium and identified using internal transcribed spacer (ITS) sequencing. The three fungi with potentially higher antagonistic activity against G. boninense, based on the percentage inhibition of radial mycelial growth (PIRG) in dual culture, were Pycnoporus sanguineus (84%), Trametes lactinea (82%) and Grammothele fuligo (81%). In addition to these three isolates, Lentinus tigrinus and a Rigidoporus sp. were also able to invade sterilized oil palm blocks previously colonized by G. boninense despite having lower PIRG values. Colonization of oil palm blocks by G. fuligo, P. sanguineus, Rigidoporus sp., T. lactinea and L. tigrinus resulted in mass losses of 32.50, 30.78, 27.64, 25.20 and 19.33%, respectively, probably due to an ability to produce one or more lignocellulolytic enzyme. Further work is needed to determine the enzymatic degradation potential of these fungi under natural conditions and their possible use in the prevention of colonization of oil palm by G. boninense.     

Cross‐infectivity of oil palm by Phytophthora spp. isolated from perennial crops in Malaysia

Bud rot disease or “Pudricion del cogollo” (PC) of oil palm is a major constraint on production in Colombia and neighbouring countries such as Brazil, Costa Rica, Ecuador, Nicaragua, Panama, Peru and Surinam. To date, there are no documented reports of Phytophthora disease of oil palm in South‐East Asia. This research, therefore, was conducted to determine the pathogenic potential of Phytophthora palmivora and Phytophthora nicotianae on oil palm using both in vitro and nursery inoculation experiments. In vitro inoculation of both P. palmivora and P. nicotianae on immature oil palm leaflets caused discoloration within 2 days of inoculation and incubation at 25 ± 1.5°C, 100% RH. Similarly, in nursery trials, lesions formed on the buds (unopened leaflets) 3 days after inoculation with P. palmivora or P. nicotianae zoospore suspensions. No lesions developed on untreated leaflets in either in vitro or nursery inoculation experiments. Phytophthora spp. were re‐isolated from leaflet lesions and confirmed as the inoculated pathogens.     

Phylogenetic and pathogenicity evaluation of the marasmioid fungus Marasmius palmivorus causing fruit bunch rot disease of oil palm

Marasmius palmivorus is a marasmioid fungal species that exhibits parasitic behaviour, although most marasmioids are rarely parasitic. The fungus has been reported to cause fruit bunch rot disease of oil palm and coconut, but only a few studies on its pathogenic behaviour are available, particularly on oil palm. Hence, there is a need to assess the ability of the fungus to act as a pathogen and to study its molecular evolution and taxonomy. Nine isolates of M. palmivorus were successfully isolated from basidiocarps and diseased fruitlets of oil palm collected from oil palm plantations and were morphologically characterized on potato dextrose agar (PDA) followed by molecular identification based on nucleotide sequence alignments of internal transcribed spacer (ITS) regions of ribosomal RNA (rRNA) gene clusters with sequences from GenBank. Koch's Postulates confirmed that M. palmivorus could infect oil palm fruitlets with symptoms similar to those observed for bunch rot disease. Molecular phylogenetic studies using nucleotide sequences of ITS and the nuclear ribosomal large subunit (nLSU) showed that isolates sharing the same phenotypic characteristics of Marasmius palmivorus and Marasmiellus palmivorus are monophyletic and share a common ancestor. The fungus has also been shown to be more closely related to the genus Marasmius than Marasmiellus; therefore, we support retention of the taxon name of the pathogen causing bunch rot disease of oil palm as Marasmius palmivorus.     

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.     

The true nature of Ganoderma in Iran: Taxonomy based on ITS and mtSSU rDNA

The genus Ganoderma Karst. has broad‐spectrum usage in biotechnology, medicine and the food industry. The complexity of the morphology within species has led to uncertain identification in the past, but recent advancements in molecular identification methods have provided scientists with the opportunity to better understand the taxonomy of the species. The present study attempts for the first time to elucidate the distinctiveness of the Ganoderma species growing in Iran concerning those elsewhere in the world based on mitochondrial small subunit ribosomal DNA (mtSSU rDNA) and internal transcribed spacer (ITS) sequence data. The results disclosed that the G. lucidum Karst. samples collected in Iran are more similar to the European Ganoderma species than to the Asian (Chinese) one used in this study.     

Wood‐rotting basidiomycetes are a minor component of fungal communities associated with Acacia hybrid trees grown for sawlogs in South Vietnam

Acacia hybrid (Acacia mangium × Acacia auriculiformis) clones are widely planted in Vietnam with a total of approximately 400,000 ha to meet the demand for pulpwood, sawn timber and wood chip exports. Silvicultural techniques such as pruning and thinning have been applied to improve productivity and sawlog quality of Acacia hybrid plantations. However, those techniques may also create opportunities for wood decay fungi to enter the Acacia hybrid stems through wounds and cause stem defects that reduce sawlog quality and the value of the plantation. The presence of fungal decay agents in Acacia hybrid trees was examined in two Vietnamese plantations. In July 2011, just prior to a second thinning, discoloured wood samples were taken from a three‐year‐old Acacia hybrid plantation at Phan Truong Hai for the isolation of fungi. In July 2012, approximately 18 months after pruning and thinning treatments, discoloured wood samples were taken from a three‐year‐old Acacia hybrid plantation at Nghia Trung for the isolation of fungi. DNA sequencing of the rDNA ITS identified the isolates. In May 2015, approximately 4 years after thinning and fertilizer treatments, discoloured and decayed wood samples were taken from the above (7‐year‐old) Acacia hybrid plantation at Phan Truong Hai for fungal identification. DNA was extracted directly from discoloured and decayed wood samples and fungal rDNA ITS amplicons sequenced on a Roche 454 sequencer. The results showed that silvicultural treatments did not affect the fungal communities associated with discoloured and decayed wood of Acacia hybrid plantation at Phan Truong Hai. A total of 135 fungal species or OTUs (operational taxonomic units) were identified, including 82 members of Ascomycota and 52 Basidiomycota.     

Oil palm survival under climate change in Kalimantan and alternative SE Asian palm oil countries with future basal stem rot assessments

Oil palm (OP) is affected by climate change and the cultivation of the crop also contributes to climate alteration. Simulation modelling and large data sets indicated the effects of climate change on OP will be to (a) reduce growth and yields and (b) cause death of some OP. Furthermore, basal stem rot (BSR) caused by Ganoderma boninense, a serious disease of OP, has increased over the past two or three decades because of the disease spreading from infection foci at a greater rate in repeated crops of OP planted on infested sites. The disease may increase further by natural selection of more virulent strains of the fungus. In this paper, increased mortality of OP is predicted based on climate change scenarios for Kalimantan and other South‐East Asian countries with palm oil industries. Climate suitability scenarios for growing OP are also used to assess how BSR will change in the specified region and countries. The data suggest that Kalimantan and the Philippines may be more suitable for growing OP than Thailand and Myanmar, with Papua New Guinea being intermediate. Development of methods for mitigating the effects of climate change on OP is urgently required.     

Environmental and stand conditions related to Fistulina hepatica heart rot attack on Castanea sativa

Castanea sativa forests in north‐eastern Spain have historically been healthy and have been good producers of timber and non‐wood forest products. In the last quarter of a century, Fistulina hepatica has gained relevance in this ecosystem and is believed to be responsible for Chestnut Red Stain (CRS), which inflicts heart rot in trees and causes severe economic losses. The fungus grows in the heartwood and produces no external symptoms that would enable early detection. With the objective of developing an inexpensive method to detect heart rot and to discover possible relationships between the severity of F. hepatica attack and the silvicultural management or site characteristics of chestnut stands, 38 pure C. sativa plots were selected, sampled and analysed for the presence of the fungus. DNA was extracted from the heartwood samples, and PCR was used to amplify target sequences with specific F. hepatica primers for non‐homologous ITS1 and ITS2 regions. Correlation analysis was used to inspect the multiple interactions among the environmental and management variables examined, and a predictive model was designed for forest managers to estimate the risk of infection. Both analyses revealed that stress variables were related to CRS severity. This result is especially relevant since temperatures are expected to rise in coming decades throughout the northern rim of the Mediterranean region, where chestnut is an important species. This study provides two tools for better managing chestnut forests in the coming years: an inexpensive and fast tool for diagnosis and a predictive model to estimate the risk of infection.     

Decline of Paulownia tomentosa caused by Trametes hirsuta in Serbia

During the monitoring of the health status of nurseries and plantations in Serbia, a decline in a 5‐year‐old Paulownia tomentosa plantation was recorded. Trees displayed symptoms of dieback, massive breaking at different positions, the appearance of decay and fungal fruitbodies at the stems. Using standard isolation methods, white colonies with cottony surfaces and regular growth were obtained from the decaying wood samples, and after the oxidation degree analysis, it was determined that these isolates belonged to Davidson's group 6, indicating white rot basidiomycetes. To identify the isolated fungus, the ITS region of one selected isolate was sequenced. Based on the morphological analysis of the obtained colonies, collected fruitbodies and ITS sequence analysis, this fungus was identified as Trametes hirsuta. This is the first report of T. hirsuta on Paulownia tomentosa in Serbia. Irregularly performed pruning as the possible infection route and the implications of these findings are discussed.     

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.     

A comparison between decay patterns of the white‐rot fungus Pleurotus ostreatus in chestnut–leaved oak (Quercus castaneifolia) shows predominantly simultaneous attack both in vivo and in vitro

In this research, we examined decay patterns occurring in Quercus castaneifolia wood under natural conditions compared with controlled decay in vivo. Pleurotus ostreatus‐infected oak wood was obtained from the Sari forests in the north of Iran. The species causing decay was verified as P. ostreatus using rDNA‐ITS sequencing of pure cultures from infected sapwood. In addition to P. ostreatus, two wood‐inhabiting Ascomycota, Trichoderma harzianum and T. lixii, were present. Mass loss in oak sapwood samples exposed to P. ostreatus for 60 days was around 10 per cent. Samples were prepared from both naturally decayed wood and wood decayed under controlled conditions and examined using microscopy. P. ostreatus was found to produce a simultaneous white‐rot decay pattern in both conditions.     

Chestnut Red Stain: Identification of the fungi associated with the costly discolouration of Castanea sativa

In Europe, fungal pathogens have reduced the overall productivity of sweet chestnut (Castanea sativa) stands and continue to threaten the economic viability of forestry operations. Chestnut Red Stain (CRS) in north‐eastern Spain, locally referred to as Roig, is capable of decreasing the market value of chestnut timber to the point of rendering chestnut coppices uneconomical. Despite its economic importance, the specific cause of this red discolouration is unknown. With the objective of verifying the presence of fungi within the symptomatic wood, and identifying the fungus or suite of fungi associated with the red stain, wood samples were collected and cultured from 37 stumps found in eight recently harvested stands in the Montseny and Montnegre‐Corredor Natural Parks. To separate the fungi associated with CRS from other species inhabiting the chestnut wood, the origin of each fungal culture was mapped in every stump. The fungi were isolated from cultures and identified by sequencing the ITS region. The results provide insight into the fungal community inhabiting chestnut wood and the potential cause of CRS; nine species were identified including two species known to cause decay in chestnut. One of them, Fistulina hepatica, appears to be a likely candidate for the causal agent of CRS. This is the first study reporting the fungi associated with CRS and opens the door to new epidemiological studies focused on F. hepatica.     

Pathogenicity of fungi associated with ash dieback symptoms of one‐year‐old Fraxinus excelsior in Montenegro

In addition to Hymenoscyphus fraxineus, two fungi identified as Diaporthe eres aff. and Fusarium sambucinum aff. were also isolated from necrotic bark lesions on declining one‐year‐old Fraxinus excelsior in a forest stand in Montenegro. To examine their involvement in ash decline, a pathogenicity test was performed using under bark inoculations on one‐year‐old Fraxinus excelsior. Hymenoscyphus fraxineus was included as comparison. All three fungal species proved highly pathogenic towards one‐year‐old seedlings although lesion sizes differed significantly between the different species. Hymenoscyphus fraxineus was most aggressive, followed by F. sambucinum aff., while D. eres aff. caused the smallest lesions. This study demonstrates for the first time the ability of isolates in the D. eres and F. sambucinum species complexes to cause decline on one‐year‐old common ash seedlings.     

Phosphorus,nitrogen and potassium nutrition on Calonectria leaf blight in eucalypt plants

Calonectria pteridis causes Calonectria leaf blight (CLB), and consequently severe defoliation in eucalypt plantations, which results in losses in wood volume. To reduce the negative impacts of this disease in eucalypt, this study aimed to assess the application of different doses and combinations of the macronutrients N, P and K on the percentage of symptomatic leaf area (SLA) and defoliation induced by the pathogen. Cuttings of a clone of Eucalyptus grandis were transplanted to pots containing soil that received different dose combinations of N, P and K, according to an incomplete factorial design. At 200 days after transplanting, leaf samples were analysed for N, P and K contents, and then, the plants were inoculated. Forty‐five days post‐inoculation, SLA and percentage of defoliation were quantified. Potassium doses above 75 mg/dm³ of soil significantly reduced SLA and defoliation. The influence of N and P on defoliation was dependent on K doses, but both reduced symptomatic leaf area. The best control of the disease, expressed by decreased defoliation and symptomatic leaf area, was achieved with leaf content of N, P and K of 9.8, 0.8 and 10.4 g per kg leaf, respectively, obtained with doses of 55, 82.5 and 143 mg/dm³of soil, respectively. Therefore, N, P and K nutrition can be a component of an integrated management programme for the control of CLB in eucalypts.     

First record of Ektaphelenchoides pini associated with Ips sexdentatus on Pinus nigra laricio in Italy

In February 2015, an unexpected windstorm downed five hectares of a European black pine Pinus nigra subsp. laricio forest formation located close to Vallombrosa, Florence (Central Italy). In the following spring, an extensive survey was conducted in the area. Felled trees, stumps and all the suitable plant material were screened for the presence of the pinewood nematode (PWN), Bursaphelenchus xylophilus, by sampling wood and bark. Bark beetles were then collected from the gallery systems on the inner side of bark samples and observed in the laboratory. The following bark beetles were morphologically identified: Ips sexdentatus, Orthotomicus erosus, O. laricis and Pityogenes bidentatusa. The dissection of Ips sexdentatus allowed the extraction of numerous nematodes that were morphologically and molecularly identified as Ektaphelenchoides pini. Conversely, only few nematode specimens were isolated from either pine bark or wood. These individuals could be only molecularly identified and belonged to an undescribed nematode taxon. Even though no PWN was recorded in the investigated sites, our survey allowed the detection of a new association between E. pini and I. sexdentatus on P. nigra.     

An agglutination test for fast and specific detection of Erwinia psidii

Dieback and wilt, caused by Erwinia psidii (Ep), is one of the most important emergent diseases of Eucalyptus spp. in Brazil. Currently, pathogen detection relies on isolation of bacteria from infected plant tissue and either identification based on morphological, physiological and biochemical tests or DNA amplification using the polymerase chain reaction (PCR), which in many cases is laborious and cumbersome. Considering the need for a simpler and more rapid, yet reliable, method for detecting the pathogen, we obtained a polyclonal antibody (anti‐Ep) and developed an agglutination test for specific detection of E. psidii. The antiserum was produced against the E. psidii strain LPF534 and tested against 101 E. psidii isolates from Eucalyptus spp.; three E. psidii isolates from Psidium guajava; 23 Ralstonia solanacearum and 18 Xanthomonas axonopodis isolates pathogenic to Eucalyptus spp.; and seven endophytic isolates from Eucalyptus spp., three of which are phylogenetically related to the genus Erwinia. Results of direct ELISA indicated that a concentration as low as 3.5 µg/ml of the anti‐Ep antibody was able to detect the E. psidii antigen and that the antibody did not cross‐reacted with other bacteria pathogenic and non‐pathogenic to Eucalyptus spp. In the agglutination test, the anti‐Ep antibody showed positive reaction with all strains of E. psidii tested whereas cross‐reaction with none of the strains that belong to other taxonomic groups was observed. The agglutination test showed a detection limit of 10⁵ colony‐forming units (CFU)/ml, and its specificity was the same as that obtained by PCR amplification using E. psidii‐specific primers. These results demonstrate that the agglutination test developed here is a useful tool for specific, fast and inexpensive detection of E. psidii although only operational on pure bacterial suspensions and not yet directly from infected tissues.     

First report on the Dutch elm disease pathogen Ophiostoma novo‐ulmi from Latvia

During 2017, Dutch elm disease was investigated on 67 Ulmus glabra and 29 Ulmus laevis samples from 28 sites in Latvia. The presence of Ophiostoma novo‐ulmi was detected in 61.2% of the samples from U. glabra and in 34.5% of the samples from U. laevis. In 11% of cases, hybrids between subsp. novo‐ulmi and subsp. americana were identified, particularly in the sites in the southern part of Latvia. This study presents the first verified record of Ophiostoma novo‐ulmi and its subspecies in Latvia.