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

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

Tissue‐specific DNA levels and hyphal growth patterns of Hymenoscyphus fraxineus in stems of naturally infected Fraxinus excelsior saplings

Ten saplings of European ash (Fraxinus excelsior L.) naturally infected by the invasive ash dieback pathogen Hymenoscyphus fraxineus were collected in Ukraine and Norway and examined for bark necrosis and extension of discoloration in sapwood and pith in a stem region. Tissue‐specific colonization profiles were determined by spatial analyses of symptomatic and visually healthy stem tissues using a H. fraxineus‐specific qPCR assay and light microscopy. Our data suggest that hyphal growth in the starch‐rich perimedullary pith is of particular importance for both axial and radial spread of H. fraxineus, but that most of its biomass accumulates in sapwood parenchyma. The study confirms the results from earlier work and presents new information that refines the current stem invasion model.     

The severity and cause of leaf spot disease of Pongamia pinnata L. and fungicidal control of the pathogen

A survey on the symptom and severity of the leaf spot disease of Pongamia pinnata L. was conducted in the nurseries of the Institute of Forestry and Environmental Sciences, University of Chittagong （IFESCU）, Bangladesh Forest Research Institute （BFRI） and Aronnak Nursery in Chittagong. The highest infection percentage and disease index were found in IFESCU nursery, followed by BFRI and the lowest was recorded in Aronnak nursery. The associated organism of leaf spot disease of P. pinnata was isolated from the diseased plant parts and the pathogenicity was established with the isolated fungus. Colletorichum gloeosprioides Penz was proved to be pathogenic. The inhibition of mycelial growth of C. gloeosporioides was observed and identified as suitable fungicides （Bavistin, Cupravite and Dithane M-45） and doses （0.05, 0.10, 0.50, 1.00, 1.50 and 2.00）. The lowest and highest mycelial growth were respectively found on Bavistin and on Cupravite at the concentration of 0.05 after 8th day of incubation. It indicates that out of the three tested fungicides, Bavistin showed most effective, followed by Diathane M-45, and Cupravite was ineffective for its very little inhibition on mycelial growth.     

Fungicide sensitivity of Dothistroma septosporum isolates in the UK

Dothistroma septosporum, a causal agent of Dothistroma needle blight (DNB), is a damaging fungal pathogen of pines that has recently started to affect native Scots pine woodlands in the UK. In addition to silvicultural methods, fungicide spraying of forest nursery stock can help prevent the spread of DNB. However, the effectiveness of modern single‐site fungicides against D. septosporum and the risk of fungicide resistance evolution remain largely unknown. In this project, we aimed to establish sensitivity profiles of D. septosporum to some widely used single‐site fungicide classes in vitro, and to determine whether fungicide resistance is already present, as this could increase the spread of D. septosporum genotypes on planting stock in native woodlands. For this purpose, we compared isolates of D. septosporum, originating from pine stands unexposed to fungicides, with isolates from nursery outbreaks, for sensitivity to a range of commonly applied fungicides. Most of the fungicides we tested were effective in vitro and we observed no significant shifts in sensitivity in forest nurseries. Although further tests in planta are required to confirm effectiveness of single‐site fungicides against D. septosporum, our results suggest that they can be successfully used in DNB control, although appropriate measures to prevent the evolution of fungicide resistance are strongly recommended.     

6种杀菌剂对不同桉树焦枯病病原菌的室内毒力测定

由丽赤壳属（Calonectria）真菌引起的桉树焦枯病是桉树世界范围内的重要病害之一。为了筛选能有效抑制丽赤壳属病原菌的杀菌剂,本研究采用6种杀菌剂（百菌清、代森锰锌、多菌灵、甲基硫菌灵、嘧菌酯、咪鲜胺）对5种桉树丽赤壳属病原菌（Ca. cerciana、Ca. crousiana、Ca. fujianensis、Ca. pauciramosa、Ca. pseudoreteaudii）通过菌丝生长法测定其室内毒力强弱。结果显示：6种杀菌剂对5种丽赤壳属病原菌均有一定的抑制作用,且差异显著。不同杀菌剂对同一丽赤壳属病原菌的抑制效果差异显著,不同丽赤壳属病原菌对同一杀菌剂的敏感性也存在差异。综合比较分析,咪鲜胺能有效发地抑制本研究中测定的丽赤壳属病原菌菌丝生长。     

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.     

Involvement of Phytophthora spp. in chestnut decline in the Black Sea region of Turkey

Chestnut blight caused by Cryphonectria parasitica is a serious disease of Castanea sativa in the Black Sea region of Turkey. During disease surveys, dieback and decline symptoms were observed on trees without apparent blight and ink disease symptoms. Black necroses, similar to those caused by Phytophthora infections, were noted on some of the chestnut coppices and saplings in one nursery in Ordu and led to an investigation into this disease complex. Only symptomatic plants showing dieback symptoms were investigated. Soil samples together with fine roots were collected from two directions, north and north‐east, approximately 150 cm away from the main stems. Phytophthora spp. were baited with young chestnut leaves. Three Phytophthora spp., P. cambivora, P. cinnamomi and P. plurivora, were identified from 12 soil samples collected from 73 locations, while from the nurseries, only P. cinnamomi was obtained. Phytophthora cinnamomi was the most common species, obtained from seven locations in five provinces and from four nurseries having similar symptoms mentioned above in different locations. Phytophthora cambivora and P. plurivora were less frequently obtained, from three to two stands, respectively. Phytophthora cinnamomi and P. cambivora were the most aggressive species when inoculated at the stem base on 3‐year‐old chestnut saplings, killing six saplings of eight inoculated in 2 months. The three Phytophthora species were first recorded on chestnut in Black sea region of Turkey with the limited samples investigated in a large area about 150 000 ha chestnut forest.     

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.     

Fraxinus excelsior seed is not a probable introduction pathway for Hymenoscyphus fraxineus

We investigated the transmission of Hymenoscyphus fraxineus from infested seed to germinating seedlings of common ash (Fraxinus excelsior) in order to determine the potential risk associated with intra‐ and intercontinental movement of seed. Neither fungal isolations from necrotic or healthy embryos nor PCR testing with H. fraxineus‐specific primers detected the pathogen. Similarly, H. fraxineus was not detected in axenically grown seedlings generated from infested seed lots. The results help clear up prior confusion of the pathogen being seed‐borne. Any remaining surface contamination by pathogen spores could be washed off seeds as a quarantine measure.     

Control of Hymenoscyphus fraxineus,the causal agent of ash dieback,using composting

Viability of Hymenoscyphus fraxineus inocula following temperature treatments for different exposure times was examined in vitro and in aerated flask‐ and large‐scale composting tests using green waste. After an exposure for up to 10 days at 20°C, 97.3% of H. fraxineus mycelium and pseudosclerotia plate cultures remained viable. No viability was detected following a 3‐day exposure to 40°C or a 1‐day exposure to 45°C although pseudosclerotia were more tolerant than mycelium to an exposure to 35°C. Primordial apothecia of H. fraxineus emerged from 62%–100% of infected ash rachises collected from two infected sites and stored at 4°C for 0–5 months; exposure to compost for up to 10 days at 20°C did not affect this emergence. No emergence of H. fraxineus apothecia was observed from ash rachises that were exposed to compost at 45°C for 1 day or at 35°C or 40°C for 3 days in flasks or at 40°C for 1 day or at 30°C for 5 days in a large‐scale composting system. Based on a fitted model, estimates of the survival of H. fraxineus inoculum in infected ash rachises exposed to compost at 50°C for 1 day were 0.081% of that in the untreated H. fraxineus ash rachis inoculum. Increasing loss in viability of H. fraxineus inoculum in infected ash rachises during longer and warmer exposures to compost at 35°C–45°C corresponded with a reduced concentration of pathogen DNA detected in the rachises using real‐time PCR. However, exposure of rachises to compost at >53°C resulted in a smaller reduction in pathogen DNA detected than exposure to compost at lower temperatures, possibly due to the inhibition of enzymatic degradation of DNA at elevated temperatures.     

Evaluation of fungicides for the control of Botryosphaeria corticola on cork oak in Catalonia (NE Spain)

Botryosphaeria canker of cork oak (Quercus suber L.), caused by Botryosphaeria corticola, is a well-known factor contributing to the decline of this forest species and is a serious disease in the main cork-producing countries of the Western Mediterranean basin. In this work, fungicides with the potential for controlling the disease were selected using a combination of in vitro tests and field trials. Fourteen fungicides in different chemical families were tested in vitro to evaluate their effect on inhibition of mycelial growth. The most effective fungicides were benomyl, carbendazim, cyprodinil + fludioxonil, thiabendazol and thiophanate-methyl; these compounds were then further tested under field conditions. In the field trials, carbendazim and thiophanate-methyl were the most effective fungicides, as shown by efficacy indices, in reducing debarked trunk surface affected by cankers (76% and 50–65%, respectively). These results suggest that the occurrence of Botryosphaeria canker on cork oak can be reduced effectively with the application of selected fungicides after cork removal from the trees.     

Action of fosetyl‐al and metalaxyl against Phytophthora austrocedri

Fosetyl‐Al and metalaxyl, the most commonly used systemic fungicides against Phytophthora, were evaluated for their efficacy to control Phytophthora austrocedri, the pathogen that causes a serious disease at the Austrocedrus chilensis forests in Patagonia. The effect of the chemicals on pathogen development in vitro and in planta was analysed. Both chemicals were shown to protect plants from the pathogen. In vitro assays showed that asexual reproduction was sensitive to both chemicals. However, mycelial growth and sexual reproduction, which were clearly sensitive to metalaxyl, were sensitive only to high concentrations of fosetyl‐Al. Fosetyl‐Al and metalaxyl had almost the same efficacy when applied preventively by soil drench to seedlings. This difference between in vitro and in planta results can be attributed to the dual action of fosetyl‐Al, not only inhibiting the pathogen but also stimulating host defence. In adult trees, preventive and curative treatments were tested, but only the fosetyl‐Al preventive treatment was effective in the assayed conditions. Interestingly, seedlings pretreated with both fungicides were less susceptible to the effectors secreted by the pathogen. Our results indicate that fosetyl‐Al and metalaxyl provide some resistance to the plant besides the fungistatic direct action on the pathogen. Further studies to elucidate a possible resistance‐inducing activity of these chemicals and the mechanisms involved are underway.     

Humic and fulvic acids against boxwood blight disease caused by Calonectria pseudonaviculata

The most recent outbreaks of boxwood blight caused by Calonectria pseudonaviculata, in Hyrcanian forests of Iran, have sparked the need for research to identify eco‐friendly fungicides for management purposes. Our study was conducted to test the efficacy of humic substances against C. pseudonaviculata under both in vitro and in vivo conditions. For this purpose, humic acids (HA) and fulvic acids (FA) extracted from tea waste compost (HAT & FAT), sawdust compost (HAW & FAW) and manure compost (HAM & FAM) were tested against hyphal growth and conidial germination of C. pseudonaviculata. In addition, EC₅₀ and MIC₅₀ were determined for all humic substances tested. While none of the substances caused complete inhibition of the fungus, they showed a range of antifungal activities. Based on the high inhibitory efficacy and low related EC₅₀ and MIC₅₀, HAT and FAT provided the highest efficacy against the pathogen. In the greenhouse, curative and protective effects of the humic treatments at 2,000 mg/L were also investigated against disease severity. Of the substances tested, FAW and FAM showed the weakest antifungal effects and HAT had the greatest curative and protective effects. These results indicate that HAT application can be used as an eco‐friendly control method for boxwood blight epidemics in the disease hotspots of Hyrcanian forests.     

Direct evidence of Hymenoscyphus fraxineus infection pathway through the petiole‐shoot junction

The symptoms of ash dieback caused by the fungus Hymenoscyphus fraxineus include wilting of the foliage followed by dieback of shoots, twigs and branches. Necroses in shoots are assumed to develop after infection through leaf petioles; however, clear evidence of this infection pathway has not yet been provided. Considering the multiple pathogen genotypes in dead ash petioles, we aimed to obtain a spatial overview of all H. fraxineus genotypes colonizing individual shoots and their corresponding petioles before leaf shedding to acquire precise information about the infection biology of H. fraxineus and its ability to cross the petiole‐shoot junction. Individual genotypes of H. fraxineus were characterized by the analysis of microsatellites using DNA extracted directly from petiole segments or cultures isolated from the segments. We detected 150 different multilocus genotypes in 10 analysed shoots and their respective petioles; the highest number of genotypes was eight for a single petiole and three for a single shoot. The genotypes of most shoot lesions were identical to particular genotypes from the proximal segments of petioles, implicating the main pathway of shoot infections. To test whether the amount of colonized substrate or intraspecific competition have an effect on successful infection, genotypes that reached the most proximal end of the petioles were scored for the number of invaded petiole segments and for the number of other H. fraxineus genotypes co‐occurring in the segments. However, the extent of colonization of the scored genotypes and intraspecific competition with other H. fraxineus strains did not influence pathogen success in entering the shoot. This study confirms that the majority of ash shoot infections are caused by genotypes of H. fraxineus originating from petioles. Compared to petioles, the frequency of shoot colonization as well as number of H. fraxineus genotypes in shoots was much lower.     

Hymenoscyphus fraxineus mycelial growth on media containing leaf extracts of different Oleaceae

We assessed the mycelial growth rate of Hymenoscyphus fraxineus, the causal agent of ash dieback, on agar media containing leaf extracts of seven common Mediterranean species of the Oleaceae (Fraxinus excelsior, F. angustifolia, F. ornus, Ligustrum vulgare, Olea europaea, Phyllirea latifolia and Syringa vulgaris). The pathogen grew on all media, but growth rates showed significant differences among media and H. fraxineus isolates. Growth rates were highest on media containing F. excelsior and F. angustifolia, intermediate on media containing O. europaea and P. latifolia and lowest on those containing F. ornus and L. vulgare.     

Side effects of some fungicides on phytoseiid mites (Acari, Phytoseiidae) in north-Italian vineyards

The effects of fungicides containing mancozeb or copper oxychloride, as principal active ingredients, on phytoseiid mites were investigated in a vineyard comprising four varieties (Prosecco, Cabernet Franc, Pinot gris and Merlot) and located in north-eastern Italy. Phytoseiid colonisation was different among the four varieties: Amblyseius andersoni and Kampimodromus aberrans were dominant on Pinot gris and Merlot, respectively, while Typhlodromus pyri was more common than the above species on Prosecco and Cabernet Franc. Applications of mancozeb fungicides significantly affected K. aberrans populations. Concerning T. pyri, a significant effect was observed on Cabernet but not on Prosecco. The effects of mancozeb fungicides on A. andersoni were less clear. The response of phytoseiids to fungicides containing mancozeb appeared to be mediated by the variety. Therefore, the choice of one or two varieties as a standard reference for field tests is recommended. These results also suggest that the side effects of fungicides on predatory mites should be studied on different phytoseiid species and, possibly, on susceptible and resistant strains in order to gain useful insights.     

油茶苗圃炭疽病原菌鉴定及抗药性

【目的】探讨中国油茶苗圃炭疽病原菌种类,揭示油茶苗圃炭疽病菌抗药性,为其防治提供依据和指导。【方法】分离油茶苗圃炭疽病菌,并对分离菌株的ApMat基因进行PCR扩增和测序,通过对序列系统发育分析鉴定炭疽病原菌种类;采用区分剂量法测定所有菌株对杀菌剂多菌灵、乙霉威、戊唑醇和咪鲜胺的抗药性。【结果】湖南、江西、海南和广东4个省份分离的95株的油茶苗圃炭疽病病原菌主要是果生炭疽菌(Colletotrichum fructicola)、暹罗炭疽菌(C.siamense)、山茶炭疽菌(C.camelliae)和胶胞炭疽菌(C.gloeosporioides)4个种,其中炭疽病菌分布范围最广、分离数量最多(83株,占总分离菌株的87.4%)。抗药性测定结果表明,对多菌灵产生抗性的菌株有31个,占总分离菌株的32.6%;对戊唑醇产生了抗性的菌株有31株,占总分离菌株的32.6%。其中对多菌灵和戊唑醇这2种杀菌剂均具有抗性的菌株有6株,占总分离菌株的6.3%;对多菌灵、乙霉威和戊唑醇3种杀菌剂均有抗性的菌株有2株,占所有分离菌株的2.1%。但所有95个菌株对咪鲜胺敏感,说明目前苗圃油茶炭疽病菌尚未对咪鲜胺产生抗药性。29个高抗多菌灵而对乙霉威敏感菌株第198位氨基酸由谷氨酸(Glu)突变为丙氨酸(Ala),2株对多菌灵和乙霉威具有双重抗药性的菌株第200位氨基酸由苯丙氨酸(Phe)突变为酪氨酸(Tyr)。【结论】我国油茶苗圃炭疽病菌已对多菌灵/乙霉威和戊唑醇产生严重的抗药性,但对咪鲜胺尚未检测到抗药性。     

Can Hymenoscyphus fraxineus infect hardy members of the Oleaceae other than ash species?

The interaction between the ash dieback fungus, Hymenoscyphus fraxineus, and two commonly grown members of the Oleaceae (Forsythia × intermedia ‘Lynwood’ and Ligustrum vulgare) was investigated. Shoots were inoculated with three isolates of H. fraxineus and harvested after 2 and 10 weeks. DNA was extracted from tissue collected at the point of inoculation, as well as 15 mm below and above (distal), and analysed using fungal‐specific ITS primers and specific primers for H. fraxineus. The fungus was frequently detected in tissue at the point of inoculation, but rarely in distal samples. There was no evidence that H. fraxineus was pathogenic on either of the two oleaceous species over the duration of the experiment.     

Local population structure of Hymenoscyphus fraxineus surveyed by an enlarged set of microsatellite markers

The population structure of Hymenoscyphus fraxineus, the causal agent of ash dieback, was assessed at four closely located sites in the Czech Republic. To analyse the genetic variation, one Swiss and one Norwegian population with known population structures were selected as reference points. The analysis was performed using 16 previously published and five newly developed microsatellite markers. The quality of the new markers was assessed by sequencing the flanking region, identifying the type of the mutation and analysing the inheritance and linkage between all pairs of loci. In addition, markers were tested on 45 Japanese strains to confirm their usability on native H. fraxineus populations in Asia. One of the new markers was monomorphic in all European populations and one marker exhibited a high percentage of null alleles in the Japanese samples. Twenty markers in the four Czech populations showed lower average gene diversity than in the other two European populations. One Czech population significantly differed from all the others, with a pairwise G_ST of approximately 0.2, the lowest allelic richness and very low average gene diversity, likely resulting from a founder effect. The amount of genetic differentiation between the four neighbouring Czech populations exceeds the expected value from previous Europe‐wide population studies and suggests that local population dynamics can affect the population structure of H. fraxineus.     

Fine root dynamics of oak saplings in response to Phytophthora cinnamomi infection under different temperatures and durations

The belowground effects of Phytophthora cinnamomi on 1‐year‐old saplings of two common oak species in mid‐Atlantic US forests, white (Quercus alba) and red oak (Q. rubra), were examined after incubation in pathogen‐infested soilless potting mix. Fine root lengths (0–1.5 mm in diameter) of both oak species were quantified after incubation at successive 30‐day intervals up to 300 days, for a total of 10 incubation periods. In addition, colony‐forming units (CFU) of P. cinnamomi were quantified after white oak saplings were incubated in infested soilless potting mix at different temperature/duration combinations that reflect soil conditions present in the mid‐Atlantic United States. Impact of P. cinnamomi on fine root lengths of red and white oak saplings varied considerably over time. Significant periods of fine root loss occurred primarily during spring, when bud break and leaf flush began for both oak species. Red oaks had 17% fine root loss on average, while white oaks appeared more resistant to P. cinnamomi infection with a 2% decrease in fine roots over the course of the experiment. Phytophthora cinnamomi CFU declined significantly with exposure to all incubation temperatures except 8°C. This was in contrast to in vitro experiments, where the optimum temperature for mycelial growth was determined to be 21°C and above. Significant fine root loss caused by P. cinnamomi depended on plant phenology and the oak species tested. Extreme soil temperatures have a significant adverse impact on temporal changes of P. cinnamomi population.