Auxin‐mediated relationships between apple plants and root inhabiting fungi: impact on root pathogens and potentialities of growth‐promoting populations

This study aimed to elucidate the relationship between plant hosts and root‐colonizing fungi recovered from apple orchard soils that had been replanted over multiple generations. Functional relationships of three groups of filamentous fungi (Ceratobasidium sp., Cylindrocarpon‐like group and Fusarium acuminatum) with apple rootstocks were evaluated in plant growth bioassays. The Cylindrocarpon‐like group and Ceratobasidium sp. showed a relationship with the host plant varying from pathogenic to commensal through to mutualistic for the latter group, while that of F. acuminatum tended to be mutualistic. Seven fungal isolates of each group, which induced the highest plant growth in bioassays, were evaluated for auxin (IAA) and gibberellin (GA₃ and GA₄) production in culture filtrate. All isolates of F. acuminatum as well as most of those of the Ceratobasidium sp. and Cylindrocarpon‐like groups produced IAA in culture filtrate. IAA production was evaluated for additional isolates of endophytic fungal species from fruit tree orchards and the functionality of IAA was confirmed by growing in vitro micropropagated plantlets of apple rootstock on MS medium supplemented with fungal culture filtrate. Findings from this study may explain the difficulty in defining the precise role of diverse root‐colonizing fungal populations in replant disease aetiology of fruit tree orchards. However, the results demonstrate the presence of a positive and widely available biotic component of the orchard soil biology that may be exploited for the benefit of tree growth and production.     

Identification of Neofabraea species causing bull's eye rot of apple in Poland and their direct detection in apple fruit using multiplex PCR

Based on partial sequence analysis of the β‐tubulin gene, 19 isolates of fungi causing bull's eye rot on apple in Poland were classified into species: Neofabraea alba, N. perennans and N. kienholzii. To the authors’ knowledge, the detection of N. kienholzii is the second in Europe and the first in Poland. Species affiliation of these fungi was confirmed by a new species‐specific multiplex PCR assay developed on the basis of previously published methods. The new protocol allowed for the specific identification of bull's eye rot‐causing species, both from pure cultures and directly from the skin of diseased or apparently healthy apples. In 550 samples of diseased fruits collected from nine cold storage rooms located in three regions of Poland, in 2011 and 2012, N. alba was detected as the predominant species causing bull's eye rot, occurring on average in 94% of the tested samples. Neofabraea perennans was found in a minority of apple samples, N. kienholzii was found only in two apple samples, while N. malicorticis was not detected in any sample tested. In tests on 120 apparently healthy fruits, only N. perennans was detected in a single sample. The results of genetic diversity analyses of bull's eye rot‐causing fungi based on the β‐tubulin gene sequence and an ISSR (inter‐simple sequence repeat) PCR assay with two primers were consistent, showing the expected segregation of tested isolates with respect to their species boundaries. However, the genetic distance between N. perennans and N. malicorticis was very low, as reported previously.     

Characterization of Glomerella cingulata isolates from almond fruit using VCG,molecular, fungicide sensitivity and pathogenicity tests 1

Almond anthracnose caused by Glomerella cingulata is a major disease of this crop in Israel. The pathogen infects young fruit resulting in fruit rot. Leaf wilting and shoot dieback accompany fruit rot, even though the pathogen cannot be isolated from leaves or twigs. Isolates of G. cingulata from diseased almond fruit were compared using vegetative compatibility grouping (VCG), molecular methods, fungicide sensitivity and pathogenicity assays in order to determine the genetic diversity and host specificity among different populations. Polymerase chain reaction amplification of genomic DNA, using four primers produced uniform banding patterns for all the almond isolates from different geographic locations in Israel. HaeIII digestion patterns of A + T-rich DNA, and Southern hybridization of the repetitive nuclear DNA element (GcpR1) to PstI-digested genomic DNA of almond isolates also revealed no polymorphism. Chlorate-resistant nitrate-nonutilizing (nit) mutants were generated and used in heterokaryon tests. Complementary heterokaryons formed between the mutants of different isolates indicated a single VCG. Isolates of G. cingulata from almond had optimal growth temperatures of 20–22°C as opposed to 26–28°C for avocado isolates. In addition, almond isolates of G. cingulata are insensitive to benzimidazole fungicides in contrast to sensitivity of isolates from avocado. In artificial inoculations, almond isolates infected almond, avocado, apple, mango and nectarine fruit at a slower rate than G. cingulata isolates from avocado, apple and mango. Only the anamorph Colletotrichum gloeosporioides has been detected on almond in Israel, whereas isolates of G. cingulata from other hosts produce ascocarps.     

A real‐time (TaqMan) PCR assay to differentiate Monilinia fructicola from other brown rot fungi of fruit crops

To prevent the entry and spread of the brown rot fungus Monilinia fructicola in Europe, a fast and reliable method for detection of this organism is essential. In this study, an automated DNA extraction method combined with a multiplex real‐time PCR based on TaqMan chemistry was developed for fast, convenient and reliable detection of both the EU quarantine organism Monilinia fructicola and the three other brown rot fungi M. fructigena, M. laxa and Monilia polystroma. Using the internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene repeat, a Monilinia genus‐specific primer pair and two differently labelled fluorogenic probes specific for M. fructicola and the group M. fructigena/M. laxa/Monilia polystroma were developed. The analytical specificity of the assay was assessed by testing 33 isolates of the four brown rot fungi and 13 isolates of related fungal species or other fungal species that can be present on stone and pome fruit. No cross‐reactions were observed. The assay was found to have a detection limit of 0·6 pg of DNA, corresponding to 27 haploid genomes or four conidia. Comparison of a manual DNA isolation followed by a conventional PCR with an automated DNA isolation combined with the presently developed real‐time PCR showed that the latter method gave improved results when tested with 72 naturally infected stone fruit samples. The detection rate increased from 65 to 97%.     

Multiple Alternaria species groups are associated with leaf blotch and fruit spot diseases of apple in Australia

Leaf blotch and fruit spot of apple caused by Alternaria species occur in apple orchards in Australia. However, there is no information on the identity of the pathogens and whether one or more Alternaria species cause both diseases in Australia. Using DNA sequencing and morphological and cultural characteristics of 51 isolates obtained from apple leaves and fruit with symptoms in Australia, Alternaria species groups associated with leaf blotch and fruit spot of apples were identified. Sequences of Alternaria allergen a1 and endopolygalacturonase gene regions revealed that multiple Alternaria species groups are associated with both diseases. Phylogenetic analysis of concatenated sequences of the two genes resulted in four clades representing A. arborescens and A. arborescens‐like isolates in clade 1, A. tenuissima/A. mali isolates in clade 2, A. alternata/A. tenuissima intermediate isolates in clade 3 and A. longipes and A. longipes‐like isolates in clade 4. The clades formed using sequence information were supported by colony characteristics and sporulation patterns. The source of the isolates in each clade included both the leaf blotch variant and the fruit spot variant of the disease. Alternaria arborescens‐like isolates were the most prevalent (47%) and occurred in all six states of Australia, while A. alternata/A. tenuissima intermediate isolates (14%) and A. tenuissima/A. mali isolates (6%) occurred mostly in Queensland and New South Wales, respectively. Implications of multiple Alternaria species groups on apples in Australia are discussed.     

A survey of entomopathogenic nematodes of the families Steinernematidae and Heterorhabditidae (Nematoda: Rhabditida) in Damascus countryside of Syria

A survey of entomopathogenic nematodes was conducted in Damascus countryside from January 2011 to December 2013. Soil samples were tested for the presence of steinernematid and heterorhabditid nematodes by baiting with Galleria mellonella larvae. Of the 189 soil samples studied 17 were positive for entomopathogenic nematodes (9%), with 11 of these positive samples (65%) containing Heterorhabditis and 6 (35%) Steinernema isolates. Morphological studies were carried out to characterize eight isolates. The Heterorhabditis isolates collected in Syria were identified as Heterorhabditis zealandica (Poinar, 1990), Heterorhabditis indica (Poinar et al., 1992) and Heterorhabditis bacteriophora (Poinar, 1990). Heterorhabditis zealandica was isolated from 4 sites. Heterorhabditis indica and H. bacteriophora were isolated from two sites each. Entomopathogenic nematodes were mainly found in stone fruit orchards and apple orchards but also in citrus groves, vineyards, and walnut orchards.     

Cross-infection of subtropical and temperate fruits byColletotrichumspecies from various hosts

Forty-two isolates ofColletotrichum gloeosporioidesfrom almond, apple, avocado, mango, pecan, and eight isolates ofC. acutatumfrom apple, peach and pecan were compared by molecular analyses and a pathogenicity assay in order to determine genetic variability and host specificity. Polymerase chain reaction (PCR) amplification of genomic DNA using four different primers andHaeIII digestion patterns of genomic DNA (A+T-rich DNA) grouped theC. acutatumisolates separately from theC. gloeosporioidesisolates. Based on arbitrarily primed PCR (ap-PCR), intraspecies similarity among the isolates ofC. acutatumandC. gloeosporioidesranged from 78 to 93% and from 0 to 38%, respectively. Similarity between the isolates ofC. acutatumandC. gloeosporioidesranged from 0 to 26.5%. A+T-rich DNA grouped theC. acutatumisolates separately from those ofC. gloeosporioides, corresponding to ap-PCR analyses. Artificial inoculations with nine representative isolates on almond, apple, avocado, mango and nectarine fruit showed a variation in levels of infection. TheC. gloeosporioidesisolates from almond grew more slowly, causing significantly smaller lesions on all inoculated fruit than the other isolates. TheC. acutatumisolates from apple and peach caused similar levels of infection on all fruit, but differed significantly from theC. gloeosporioidesisolate from apple. Variation in lesion size was also observed with isolates ofC. gloeosporioidesfrom apple, avocado and mango for most fruit inoculations.     

Comparative effects of temperature and humidity on the activity of three potential antagonists of rose powdery mildew

Three reported antagonists of cucumber powdery mildew,Stephanoascus flocculosus, Stephanoascus rugulosus, andTilletiopsis washingtonensis, were tested and compared under different environmental conditions for their potential for controlling rose powdery mildew, caused bySphaerotheca pannosa var.rosae. Under controlled conditions in vitro, all three fungi induced a rapid collapse of conidia, conidiophores and hyphae ofS. pannosa var.rosae on detached leaflets of miniature roses within 48 h following their application, as observed under a SEM. Both temperature and relative humidity (r.h.) affected the activity of the antagonists differently. The colonization of powdery mildew was maximal at 26 °C, especially forSt. rugulosus andT. washingtonensis. Maximal colonization was achieved at the highest r.h. tested (90%) for all three antagonists but onlySt. flocculosus maintained a colonization of 80% or better under lower r.h. These observations stress the importance of considering environmental conditions when assessing the activity of antagonistic microorganisms.     

Role of Cylindrosporium-type conidia of Mycosphaerella pomi (Pass.) Lindau: cause of Brooks fruit spot of apple, as an infection source in apple orchards

Ascospores of Mycosphaerella pomi, the pathogen of Brooks fruit spot of apple, were produced in pseudothecia on previously infected and overwintered apple leaves from late April through early August in Aomori Prefecture, Japan. In June 2003, the ascospores were germinating and producing Cylindrosporium-type conidia on apple fruit and leaf surfaces in an orchard. After ascospores were sprayed on apple leaves, Cylindrosporium-type conidia developed on the leaf surfaces. Such Cylindrosporium-type conidia caused typical symptoms of Brooks fruit spot on apple trees after inoculations. These results suggested that the Cylindrosporium-type conidia also serve as an infection source, in addition to the ascospores, for Brooks fruit spot in apple orchards.     

Testing variability in pathogenicity ofPhytophthora cactorum, P. citrophthora andP. syringae to apple, pear, peach, cherry and plum rootstocks

The relative virulence ofPhytophthora cactorum andP. syringae originating from almond trees, and ofP. citrophthora originating from citrus, to apple, pear, peach, cherry and plum rootstocks, was studiedin vivo andin vitro. Results of the different experiments were in good agreement. All testedPhytophthora isolates showed little virulence to pear rootstocks-causing only minor crown rot symptoms - and no virulence at all to apple rootstocks. In contrast, they were highly virulent to stone fruit rootstocks, causing crown rot disease. The non-pathogenicity of these isolates to pome rootstocks could be interpreted as strict host specificity.     

梨和苹果种质对阿太菌果腐病菌的抗性评价及其防治药剂筛选

为明确不同梨和苹果种质对阿太菌果腐病菌Athelia bombacina的抗性以及筛选防治其有效杀菌剂,采用离体菌丝块有伤接种方法对40份梨种质和154份苹果种质进行病斑直径测定,通过聚类分析法和平均病斑直径法对不同种质进行了抗病性分级,并利用菌丝生长速率法测定了13种常用杀菌剂对阿太菌果腐病菌的毒力。结果表明,根据聚类分析法和平均病斑直径法均可将梨和苹果种质划分为高抗、抗、中抗、感和高感5类。与平均病斑直径法相比,梨和苹果种质分别以欧式距离为14和10作为最佳聚类分割点时进行聚类分析能够更加科学地划分类别,从40份梨种质中筛选出金锤子梨1个高抗种质,仅占总数的2.50%,从154份苹果种质中筛选出垂丝海棠、莫斯科透明、新疆苹果、路边石、伏帅等32个高抗种质,占总数的22.73%。药剂试验结果表明,戊唑醇、腈菌唑、咯菌腈和噻呋酰胺对阿太菌果腐病菌菌丝生长的抑制效果较好,抑制中浓度EC_(50)分别为0.027、0.048、0.054和0.095 mg/L。表明梨和苹果种质均可被阿太菌果腐病菌侵染,但不同种质间抗病性差异明显,戊唑醇是采前防治阿太菌果腐病菌菌丝生长的最佳药剂。     

Viability and release of Neonectria ditissima ascospores on apple fruit in Brazil

During European canker monitoring in an apple experimental orchard, 14 mummified fruit (two and three trees with 10 and four positive records in 2018 and 2019, respectively) showed perithecia. Perithecium production on apple fruit, confirmation of pathogenicity of Neonectria ditissima isolated from mummified fruit, and ascospore release from fruit tissues has rarely been reported, and their role in the epidemiology of European canker has been largely overlooked. Thus, the objectives of our study were to (a) prove the presence of both conidia and ascospores of N. ditissima in mummified fruit in an experimental field, confirming pathogenesis in different apple cultivars, and (b) monitor production of the two types of inoculum in infected apple fruit over time. Canker incidence in this orchard was 47% of trees with symptoms in 2018 and 48% in 2019. Molecular and morphological tests confirmed that the fungus detected in the mummified apple fruit was N. ditissima. Apple fruit with sporodochia and perithecia washed immediately after collection from the orchard showed conidia but no ascospores of N. ditissima. However, after 4 days’ incubation, perithecia on mummified fruit showed many ascospore cirri. Koch's postulates were fulfilled on apple plants and mature fruit. Fruit inoculated with N. ditissima released spores for over a year under Brazilian field conditions. The release of both spore types peaked in May (Brazilian leaf fall) and October (spring); release of conidia also peaked in February (early harvest). These results support our hypothesis that fruit can serve as primary inoculum for European canker in Brazilian apple orchards.     

Diversity and pathogenicity of the Ceratocystidaceae associated with cacao agroforests in Cameroon

Knowledge of the diversity and ecology of plant pathogenic fungi in cacao agroforests and surrounding natural ecosystems can inform the development of sustainable management strategies for new cacao disease outbreaks. This study investigated the occurrence of fungi related to the Ceratocystidaceae and their nitidulid beetle vectors in cacao agroforests in Cameroon, under diverse agroecological conditions. The fungi and their vectors were collected from artificially induced stem wounds on cacao and associated shade trees. Collections were also made from abandoned cacao pod husks and other tree wounds within and around plantations. Fungal isolates were identified using DNA sequence‐based phylogenies and morphological comparisons, and two representatives of each species were evaluated for pathogenicity on cacao. Five species of Ceratocystidaceae were recovered, including Huntiella chlamydoformis sp. nov., H. pycnanthi sp. nov. and H. moniliformis, as well as Thielaviopsis cerberus and T. ethacetica. The incidence of these fungi appeared to be influenced by the prevailing agroecological conditions. Nitidulid beetles in the genus Brachypeplus were found to be their most common insect associates on cacao. Both T. ethacetica and H. pycnanthi produced extensive lesions after inoculation on branches of mature cacao trees, while T. ethacetica also caused pod rot. Although their impact remains unknown, fungi in the Ceratocystidaceae and their nitidulid beetle vectors are common and probably contribute to the parasitic pressure in Cameroonian cacao agrosystems.     

The threat of wild habitat to scab resistant apple cultivars

Evaluations of plant resistance to pathogens are rarely made using isolates from wild habitats, although the heterogeneity of such habitats may generate pathogen diversity which could be a source of new virulence in cultivated habitats. The aim of this study was to investigate whether scab resistance factors, identified and characterized in apples using isolates of Venturia inaequalis from a cultivated habitat, remained effective against isolates from a wild habitat. Three V. inaequalis core collections originating from the cultivated apple Malus × domestica and from two wild species, M. sieversii and M. sylvestris, were established to maximize pathogen diversity. For each core collection, 10 isolates were inoculated in mixtures onto 51 genotypes from an apple progeny segregating for two qualitative resistance genes and six quantitative resistance loci (QRL). On each apple genotype, isolates that contributed to the scab symptoms were identified within the mixture using microsatellite markers. The most frequently detected isolates were inoculated singly to compare their aggressiveness according to their host origin. The results showed that isolates from a wild habitat were able to infect the susceptible apple genotypes. However, these isolates were never more aggressive than isolates from the cultivated habitat on the resistance factors tested. It can therefore be concluded that the resistance factors used in this study, identified with V. inaequalis isolates from a cultivated habitat, remained effective against isolates from M. sylvestris and M. sieversii.     

Characteristic of Monilinia spp. fungi causing brown rot of pome and stone fruits in Poland

Brown rot, caused by fungi belonging to the genus Monilinia, is one of the most important diseases of stone and pome trees in the world. During the summers of 2010 and 2011, a total of 670 Monilinia spp. isolates were obtained from infected fruits. They were collected from different commercial stone and pome fruit orchards, located in northern, southern and central Poland. All isolates were identified using multiplex PCR. Twenty isolates obtained from plum, peach and apple fruits were identified as M. polystroma and 5 isolates from plums as M. fructicola. The remaining isolates were identified as M. fructigena or M. laxa. The identification of the isolates was also confirmed on the basis of growth characteristics in culture according to the EPPO standard PM 7/18. A comparison of morphological features of four Monilinia spp. growing on two selective growth media, APDA-F500 and CHA, indicated significant differences between these species. In artificial inoculation of fruits, all the examined Monilinia spp. isolates were pathogenic. The species affiliation of M. polystroma and M. fructicola isolates collected from orchards in Poland was confirmed on the base of phylogenetic and sequence analysis of the internal transcribed spacer (ITS1/5.8S rDNA/ITS2) region of ribosomal DNA.     

Factors associated with the distribution of some phylloplane microbes

Using the spore-fall method, colonies ofSporobolomyces andTilletiopsis were isolated, during autumn, from leaves of ferns, conifers, mono- and di-cotyledonous plants. Colonies on agar, which mirror-imaged leaf surface distributions, indicated that the above-mentioned fungi were, on some hosts, restricted to leaf margins while on others they occurred mainly along veins, were absent from veins or were randomly distributed. Colonies were commonly more numerous on parasitized than on undamaged leaves. Increased numbers ofSporobolomyces were associated with rust and powdery mildew attacks and damage by (a) the nematodeAphelenchoides ritzema-bosi and (b) the miteEriophyes macrorrhynchus. Only powdery mildews increased significantly the incidene ofTilletiopsis.Samenvatting Gebruik makend van de sporeval-methode werden gedurende de herfst kolonies vanSporobolomyces enTilletiopsis geïsoleerd van bladeren van varens, coniferen, en mono- en dicotyle planten. De verdeling van de kolonies op agar, die zich naar het spiegelbeeld van de verspreiding op het bladoppervlak ontwikkelde, gaf aan dat beide schimmels zich op sommige waardplanten beperkten tot de bladranden, terwijl zij zich op andere voornamelijk langs de nerven bevonden, niet bij de nerven voorkwamen, of willekeurig verspreid waren.De kolonies waren gewoonlijk talrijker op aangetaste bladeren dan op onbeschadigde bladeren. De toename vanSporobolomyces ging samen met roest- en meeldauwaantastingen en schade door (a) de nematodeAphelenchoides rtizema-bosi en (b) de mijtEriophyes macrorrhynchus. Een belangrijke toename vanTilletiopsis werd alleen door meeldauwen verrorzaakt.     

Characterization ofCryphonectria parasitica isolates collected from Aydın province in Turkey

The major chestnut-growing areas in Aydın Province, which yield nearly 35% of Turkey’s production, were examined for the presence of chestnut blight and bark samples were collected from the canker formations. Generally virulent type cankers were observed. Of 31 villages, 22 were found to be contaminated with the disease. A total of 97Cryphonectria parasitica (Murrill) M.E. Barr. isolates were characterized for the presence of potential hypovirulence by looking at cultural characteristics on media and virulence in cv. ‘Golden Delicious’ apple fruit. The isolates were placed into four groups based on pigmentation and abundance of pycnidia formation. Isolates produced lesions of various sizes on apple fruits but lesion size was not correlated with cultural characteristics typical to hypovirulent isolates. Among 97 isolates, ten possessing the characteristics associated with hypovirulence were assayed for the presence of dsRNA. Among these isolates only one, coded M1-3, was found to contain dsRNA. No dsRNA was detected in the single conidium isolates derived from M1-3. All of the isolates were screened for vegetative compatibility and two European vc types, EU-1 and EU-12, were detected. This low vc-type diversity in the region may indicate a high potential for spread of transmissible hypovirulence. http://www.phytoparasitica.org posting May 30, 2008.     

生物菌肥和钾肥配施对苹果钾素吸收及果实品质的影响

为了研究生物菌肥和钾肥对苹果果实品质及生长发育的协同作用,以7 a生'瓦里短枝'(Vallee spur Del)为研究对象,设置T1(K2O 420 kg·hm-2)、T2(生物菌肥2520 kg·hm-2)、T3(生物菌肥2520 kg·hm-2+K2O 294 kg·hm-2)、T4(生物菌肥2520 kg·hm...