Variation of endophytic cork oak‐associated fungal communities in relation to plant health and water stress

The main objective of this study was to obtain more comprehensive knowledge about the effect of water stress on endophytic fungal communities in asymptomatic and declining cork oak trees. Six asymptomatic and six declining cork oak trees were randomly selected in a natural cork oak forest located in Sardinia, Italy. In February 2003, the soil around three asymptomatic and three declining trees was covered with a circular plastic film to reduce rain water supply with the intention to induce water stress. The remaining six trees served as controls. Predawn xylematic water potential (PWP) was used as water status indicator and measured seasonally. Between July 2003 and June 2004, fungal endophytes were isolated every 2–4 months from twigs, branches and woody tissues. Significant differences in PWP between covered and control trees were detected mainly in autumn. Gas exchange was greatest in asymptomatic control plants. All tissues were colonized by endophytic fungi. Nineteen fungal species were isolated from 1620 plant fragments. Biscogniauxia mediterranea was the most frequently isolated fungus. Its isolation frequency was significantly higher in declining covered trees than in control trees (p < 0.05). Presence of this fungus in asymptomatic control trees was significantly higher in winter than in summer. Water stress seems to reduce species diversity of the endophytic mycobiota in cork oak and to promote proliferation of some potentially pathogenic endophytes.     

Discula quercina as a possible causal agent of dieback on oak trees in Iran

During spring and summer of 2016, dieback symptoms including blights of leaves, twigs, and acorns were observed on current year shoots of Quercus infectoria in the Zagros oak forests of Iran. The fungus isolated from damaged tissues was identified as Discula quercina based on both morphological characteristics and ITS sequencing. To fulfil the Koch's postulates, a representative isolate was inoculated onto shoots of Q. infectoria and Quercus libani in both laboratory and forest conditions. Responses of the two oak species to inoculation with D. quercina were examined under laboratory and forest conditions in a completely randomized experiment. Discula quercina was clearly involved in oak dieback, and Q. infectoria was more susceptible than Q. libani to damage by the pathogen. This is the first record of the occurrence and pathogenicity of the fungus Discula quercina on Quercus infectoria. The fungus is considered as an emerging pathogen on oak trees in Zagros forests in Iran. Furthermore, the pathogenicity of the Discula quercina on Q. libani under laboratory and forest conditions increases the potential importance of this pathogen in Zagros forests.     

Incidence of Phytophthora species in oak forests in Austria and their possible involvement in oak decline

A survey on the occurrence of Phytophthora species in oak ecosystems in Austria was conducted from April to May 1999 and in June 2000. The investigations were carried out at 35 study sites distributed throughout the zone of oak forests in eastern Austria. Four oak species, including Quercus robur, Q. petraea, Q. cerris and Q. pubescens were considered in the survey. Rhizosphere soil samples were taken from sample trees, which consisted of healthy and declining trees as indicated by their crown transparency. Young oak leaflets were used as baits to recover Phytophthora species. The assemblage of Phytophthora spp. detected in Austrian oak forests consisted of five species, including Phytophthora quercina, P. citricola, P. gonapodyides, P. europaea and P. syringae. P. quercina and P. citricola were isolated from 11 and seven sites, respectively, and were thus the most common and most widely distributed species. The three other species were recovered only sporadically. P. citricola could be separated into two morphologically and genetically well‐characterized types (A and B). Phytophthora species, in particular the common P. quercina and P. citricola occurred on sites showing a wide variety of soil types, soil textures and moisture classes. There was mild evidence for connection between deteriorating crown status and the presence of Phytophthora spp. Furthermore, significant differences in contents of magnesium, as well as calcium, aluminium, nitrogen and carbon at different soil depths (0–10, 10–20 and 20–40 cm) were detected between Phytophthora‐infested and Phytophthora‐free sites. The results of the present study provide circumstantial evidence that Phytophthora species are involved in oak decline at certain sites in Austria.     

Temporal variations in endophytic fungal assemblages of Ginkgo biloba L.

We isolated endophytic fungi from living healthy leaves, petioles, and current-year twigs of Ginkgo biloba L. from April to November 2004 with the objective of identifying the dominant endophytic fungal taxa, and monitoring their occurrence and frequency. A total of 9 fungal taxa were identified to the genus level. Diversity measures inferred from the Shannon–Wiener, Morisita–Horn, and S?rensen indices indicated that leaves and petioles harbored more diverse endophytic fungal assemblages than twigs, and that fungal taxa involved in twigs shared less with those in leaves and petioles. Among the organs, the occurrence pattern of overall endophytic fungi differed significantly, and two taxa, Phomopsis sp. and Phyllosticta sp., were the most frequently isolated and thus regarded as the dominant endophytic fungi. Phomopsis sp. was isolated frequently from twigs (84.8%) but rather few from leaves (16.1%) and petioles (24.3%). Phyllosticta sp. was isolated frequently from leaves (72.9%) and petioles (65.7%) but was never isolated from twigs. Temporal changes in relative frequency of total endophytic fungi tended to differ among sampling dates for all three organs. The occurrence of Phyllosticta sp. in both leaves and petioles was first detected in August and peaked in October. Phomopsis sp. was detected in twigs throughout the growing season. These results suggest that the distribution of the two dominant endophytic fungi was organ-specific and differed within seasons.     

First records of soilborne Phytophthora species in Swedish oak forests

Thirty‐two oak stands in southern Sweden, 27 with predominantly declining trees and five with a higher proportion of healthy trees were investigated regarding the presence of soilborne Phytophthora species. Phytophthora quercina, an oak‐specific fine root pathogen, was isolated from rhizosphere soil samples in 10 of the 27 declining stands. Additionally, P. cactorum and P. cambivora were recovered from one stand each. No Phytophthora species were isolated from the healthy oak stands. The soil conditions at the sites from which Phytophthora spp. were recovered ranged from mesic sediments to moraines, with clayey to silty textures and with soil pH (BaCl₂) between 3.5 and 5.0. The results show that P. quercina is geographically widespread in oak stands in southern Sweden and indicate that this pathogen may be one of the factors involved in oak decline in Northern Europe as has already been shown for western, Central and parts of southern Europe.     

Occurrence of Phytophthora species in oak stands in Italy and their association with declining oak trees

Soil‐borne species of Phytophthora were isolated from 19 of 30 examined oak forest areas in Italy. The frequency of isolated Phytophthora spp. (35.2%) was significantly correlated with soil pH and longitude of the sites. Eleven Phytophthora species were detected. Phytophthora cambivora, P. cinnamomi and P. cactorum were recovered from sites in central and southern Italy whereas P. quercina was isolated in the northern and central part of the country. Phytophthora citricola occurred all over Italy. Phytophthora quercina was the only species significantly associated with declining oak trees.     

Endophytische Pilze in Blättern und Zweigen von Quercus petraea

Endophytic fungi in leaves and twigs of Quercus petraea . Surface sterilized leaf and twig samples taken in 1989 from sessile oak (Quercus petraea) in Austria yielded host-specific as well as unspecific ubiquistic endophytes. In leaves, Aureobasidium apocryptum and Apiognomonia quercina were the most frequent taxa, while Colpoma quercinum was the dominant species in twigs. The colonization rates of individual species were subjected to seasonal variation.     

The effect of bagging branches on levels of endophytic fungal infection in Japanese beech leaves

The species composition of the endophytic mycobiota in leaves of Japanese beech trees (Fagus crenata) and the sources for leaf infections were studied in a forest reserve situated in central eastern Honshu, Japan. To clarify the mechanism of infection of leaves, half of the branches were covered with polyethylene bags and species composition and levels of endophytic fungal infection were then compared with those of unbagged controls. Isolations were carried out from the leaves, petioles, and current‐year twigs of both, bagged and unbagged branches. Additionally, species composition was detected in overwintered terminal buds of beech trees and in the leaves of potted seedlings that had been placed in the field in different seasons. The species assemblage of the unbagged leaves, petioles, and current‐year twigs was dominated by Mycosphaerella buna, Ascochyta fagi, Periconiella sp., and Tritirachium sp. Other frequently recovered species were Xylaria sp., Phomopsis sp., and Tubakia dryina. Mycosphaerella buna and A. fagi were never isolated from leaves on bagged branches. A. fagi was, however, detected on both bagged and unbagged petioles and current‐year twigs at comparatively low isolation frequencies. The detection of Periconiella sp. on all occasions in both bagged and unbagged leaves was a characteristic feature that differs from those of the other three dominant endophytic fungi. The fungus was also detected without significant differences in bagged and unbagged petioles and current‐year twigs on most sampling dates. Furthermore, Periconiella sp. was isolated from immature twigs inside the bud scales. Tritirachium sp. was frequently detected in unbagged leaves and petioles and in both bagged and unbagged current‐year twigs, and rarely in bagged leaves and petioles, but was never recovered from terminal buds. The results of the potted seedling experiments revealed that all four dominant species had airborne inocula. The infection of leaves by M. buna occurs exclusively by airborne propagules, i.e. ascospores in spring and conidia in autumn. In Periconiella sp. hyphal growth of the fungus from immature twigs inside the buds into the leaf tissues was suggested in addition to infection by airborne inocula. Tritirachium sp. hyphae were suggested to grow from previous‐ to current‐year twigs. Ascochyta fagi was present in the outermost scales of overwintered terminal buds, but no systemic growth of the fungus into the petioles and current‐year twigs was observed. Our technique of covering the branches before new leaves unfolded was effective in preventing infection by airborne inocula of endophytic fungi.     

Geographical and seasonal influences on the distribution of fungal endophytes in Laurus nobilis

The species composition of the endophytic mycobiota in bark, leaves and twigs of Laurus nobilis were studied in Western Anatolia. Four sampling sites were selected; one of which was sampled twice, in spring and autumn. About 3892 fungal isolates obtained grouped into 85 morphological types. The 11 dominant species, with isolation frequencies >1.5%, were Alternaria alternata, Botryosphaeria sarmentorum, Colletotrichum dematium, C. gloeosporioides, Hendersonula sp., Paraconiothyrium sp., Phoma sp., three species of Phomopsis and Seimatosporium lichenicola. Numbers of fungal species isolated per tree differed between the sampled sites. Isolation frequencies of the dominant species, and other less frequent species, were dependent on the sampling site. The degree of endophytic infection and the diversity of fungal species were significantly higher in spring. Frequencies of all dominant species depended significantly on season, except for Stemphylium sp. and S. lichenicola. Analysis of the total endophytic mycoflora of the sampled trees suggested that geographical factors affects the endophytic distribution patterns more significantly than seasonal factors.     

Phytotoxins produced by the oak pathogen Discula quercina

Two phytotoxic metabolites, p‐hydroxybenzaldehyde and indol‐3‐aldehyde, were isolated and identified by spectroscopic methods from solid cultures of Discula quercina, an endophytic fungal pathogen frequently associated with oak decline in Italy. In addition, the fungus produced an unusual acyclic keto acid as a major metabolite, which was identified by spectroscopic and chemical methods as 5‐oxo‐6E,8E‐octadecadienoic acid. In leaf puncture assays on Quercus suber and Q. ilex leaves, indol‐3‐aldehyde proved to be more toxic than p‐hydroxybenzaldehyde, while 5‐oxo‐6E,8E‐octadecadienoic acid was inactive even at the highest concentration used (1 mg ml^?1). Although indol‐3‐aldehyde is a known microbial metabolite, its phytotoxic activity has hitherto not been reported. This report is the first to describe the production of phytotoxic compounds by D. quercina.     

Endophytische Pilze in Blättern und Ästen gesunder und geschädigter Buchen (Fagus sylvatica L.)

Endophytic fungi in leaves and twigs of healthy and diseased beech trees (Fagus sylvatica L.). Endophytic fungi were isolated from surface-sterilized leaves and twigs of healthy and diseased trees. Three fungal species could be isolated frequently: Apiognomonia errabunda, Diaporthe eres and Bisporella sp. D. eres occurred in leaves of healthy trees more frequently, than in those of diseased trees. It has been hypothesized, that less capacity of water and nutrient uptake of diseased trees is responsible for this effect. A mutualistic symbiosis of A. errabunda and D. eres with beech trees is supposed and discussed.     

Relative susceptibility of oaks to seven species of Phytophthora isolated from oak forest soils

Isolates of Phytophthora cambivora, P. cinnamomi, P. citricola, P. europaea, P. quercetorum and two unidentified species were tested for their pathogenicity to eastern US oak species by root and stem inoculations. Experiments were conducted during two different periods and included 1‐, 2‐ and 20‐year‐old oaks grown under greenhouse and field conditions. Species of Phytophthora were pathogenic in varying degrees to the oak species tested. All species were pathogenic to fine and taproots of at least one oak species. The fine root damage caused by the species of Phytophthora ranged from 9 to 55% when compared to the controls. Roots were more susceptible during the fall inoculation period than the summer. With exception of Phytophthora sp1 and P. quercina‐like, all species of Phytophthora were pathogenic to oak stems with P. cinnamomi and P. citricola being the most aggressive. Quercus montana and Q. rubra were the most susceptible oak species to stem inoculation. Lesion sizes were considerably larger when 20‐year‐old trees were inoculated. Generally, no significant differences in lesion sizes were detected in greenhouse tests when the summer and fall inoculation periods were compared. However, on 2‐year‐old field‐grown seedlings, lesion sizes were considerably smaller or not significantly different from controls during the fall inoculation period, suggesting lower, late season temperatures may restrict lesion development.     

Previously unrecorded low‐temperature Phytophthora species associated with Quercus decline in a Mediterranean forest in eastern Spain

Oak decline has been a serious problem in Europe since the beginning of the twentieth century. In south‐west Spain, Quercus ilex and Q. suber are the main affected species, and their decline has been associated with Phytophthora cinnamomi. During the last 10 years, a severe decline of Q. ilex and Q. faginea accompanied by a significant decrease in the production of acorns affecting natural regeneration was observed in the eastern part of the Iberian Peninsula. Therefore, the aim of this study was to investigate the possible involvement of Phytophthora spp. in the decline. A forest in the Natural Park ‘Carrascar de la Font Roja’ in Comunidad Valenciana (eastern Spain), which is dominated by Q. ilex and Q. faginea, was surveyed during 2010–2011. Symptomatic trees showed thinning and dieback of the crown, withering of leaves and death. An extensive loss of both lateral small woody roots and fine roots and callusing or open cankers on suberized roots were observed. Soil samples containing fine roots were baited using both Q. robur leaves and apple fruits. Six Phytophthora species were isolated: P. cryptogea, P. gonapodyides, P. megasperma, P. quercina, P. psychrophila and P. syringae. These are the first records of P. quercina and P. psychrophila on Q. faginea, of P. quercina in Spain and of P. psychrophila in mainland Spain. A soil infestation trial was conducted for 6 months under controlled conditions with 1‐year‐old seedlings of Q. ilex and Q. faginea. Phytophthora cinnamomi was included in the pathogenicity test for comparison. The results showed that Q. ilex seedlings were generally more susceptible to infection than Q. faginea with P. cinnamomi being the most aggressive pathogen to both oak species. The two most commonly isolated Phytophthora species, P. quercina and P. psychrophila, also proved their pathogenicity towards both Q. ilex and Q. faginea.     

Pathogenicity of fungi isolated from Quercus suber in Catalonia (NE Spain)

Summary Thirty‐four fungal species isolated from cork oak (Quercus suber) in Catalonia (NE Spain) during 1992–95 were tested for pathogenicity either in stem, leaf or root inoculations. Eleven species were found to be pathogenic on stem: Biscogniauxia mediterranea, Botryosphaeria stevensii, Diatrype cf. stigma, Endothia gyrosa, Fusarium solani, Graphium sp., Ophiostoma quercus, Phomopsis sp., Phytophthora cinnamomi, Sporendocladia bactrospora and an unidentified Coelomycete. Three fungi showed pathogenic effects on leaves: Dendrophoma myriadea, Lembosia quercina and Phomopsis quercella. No clear pathogenic effects were detected in the root inoculation experiment. Trunk pathogens were differentiated into two groups according to the effects induced in the inoculated plants; B. stevensii, Phomopsis sp. and P. cinnamomi caused the death of the inoculated plants and induced the formation of large cankers and vascular necroses. The other pathogenic species also produced severe cankers and vascular lesions, but no significant mortality was detected. Water stress increased the lesions caused by B. mediterranea and Phomopsis sp., but limited those of P. cinnamomi and the rest of the inoculated fungi. However, water stress did not significantly affect the damage caused by B. stevensii, which was the most virulent of the species tested. Leaf pathogens only showed their effects if the leaf cuticle was previously damaged. Lembosia quercina caused small dark lesions whereas D. myriadea and P. quercella produced large necrotic areas in well‐watered plants. The lesions caused by the last two fungi were reduced by water stress.     

Fungal endophytes associated with Sonneratia (Sonneratiaceae) mangrove plants on the south coast of China

The aim of this study was to describe endophytic fungal communities residing in five mangrove species (Sonneratia caseolaris, Sonneratia hainanensis, Sonneratia ovata, Sonneratia Paracaseolaris, Sonneratia apetala) collected at the south coast of China. The colonization frequency (CF) of endophytes in roots, stems and leaves was measured and the structures of endophytic communities were investigated, using endophytic fungal diversity indices and similarity coefficients. We found high biodiversity and tissue specificity of endophytic fungi. A total of 391 fungal isolates were identified using morphological and molecular methods. We recovered a total of 39 distinct endophytic species, of which Cytospora, Diaporthe, Fusarium, Glomerella, Mycosphaerella, Phoma, Phomopsis and Stemphylium were the dominant fungal taxa. Most recovered species were found on more than one host species but the dominant species differed according to host and tissue type.     

Occurrence of Phytophthora cinnamomi in cork oak forests in Italy

An increasing decline and mortality of cork oak trees have been recently observed in central Italy and Sardinia Island. Following surveys conducted in three declining cork oak forests, a Phytophthora species was consistently isolated from soil samples collected from trees displaying different level of decline. Based on morphological features, growth rates at different temperatures and analysis of DNA sequences of the ITS region, all isolates were identified as Phytophthora cinnamomi Rands. This pathogen caused large brownish lesions on inoculated freshly cut branches of cork oak. It was re‐isolated from all infected tissues. These findings represent the first report of P. cinnamomi on cork oak trees in Italy.     

A new method to detect Lonsdalea quercina in infected plant tissues by real‐time PCR

Presymptomatic and accurate diagnoses of pathogens are essential for disease prediction and the timely application of bactericide. The bacterium Lonsdalea quercina (=Brenneria quercina) has been reported as the causal agent of drippy nut and bark canker disease on oak in California (US) and Europe. In recent years, it is also found on Populus × euramericana trees in Henan province of China. This bacterium causes longitudinal cankers of a few centimetres in size on the bark surface of the upper trunk. In this study, we developed two species‐specific PCR assays using primer pairs LqfF/LqfR and LqgF/LqgR for the rapid and accurate detection of the pathogenic bacteria in diseased plant tissues. The results show that the LqfF/LqfR primers amplified only a single PCR band of approximately 382 bp and the LqgF/LqgR primers yielded a PCR product of approximately 286 bp. The two primers were successfully adapted to real‐time PCR based on SYBR Green I used with the ABI 7500 system. The detection limit of the reaction was 0.1 pg genomic DNA per 20 μl PCR reaction volumes. The pathogen was mainly detected in the phloem of cankers as well as in the exudates of diseased trees, but was not found in the xylem or leaves. The size of pathogen in distribution was larger than the lesion. The results demonstrate that real‐time PCR assays can be used to detect the pathogen by extracting DNA directly from infected plant tissues. This method is a rapid, reliable method for the presymptomatic and accurate detection of L. quercina, providing a useful insight into epidemiological studies.     

Variations in virulence and hyphal growth of four Raffaelea quercivora isolates within Quercus crispula

Mass mortality of fagaceous trees caused by Japanese oak wilt has occurred widely in Japan. Although virulence of the causal fungus, Raffaelea quercivora, appeared to differ among isolates, its relation to the fungal growth within trees was unknown. To clarify the differences in fungal virulence against susceptible Quercus crispula, we examined fungal growth of four R. quercivora isolates within trees and the resulting virulence. In our study, the isolates were multiple‐inoculated in seedlings and single‐inoculated in twigs of mature trees. In the multiple‐inoculation test, mortality rates were examined by the observation of external symptoms. In the single‐inoculation test, water conductance and hyphal growth within the trees were examined by applying aqueous dyes and fluorescence microscopy, respectively. Mortality rates, the proportion of the cross‐sectional area comprising non‐conductive sapwood and horizontal hyphal growth differed significantly among the isolates. Univariate logistic regression analyses showed that both the proportion of non‐conductive sapwood and hyphal growth were significantly positively related to mortality rates. For three isolates, hyphal growth was significantly positively correlated with the proportion of non‐conductive sapwood. These results suggested that the virulence against Q. crispula varies among R. quercivora isolates and that the extent of fungal colonization of the tree determines fungal virulence.     

Seasonal,tissue and age influences on frequency and biodiversity of endophytic fungi of Citrus sinensis in Iran

A total of 4,009 endophytic fungal isolates were recovered from healthy leaves, bark and xylem of Citrus sinensis in different seasons and age‐classes. Corresponding to those factors, the majorities of fungal isolates were recovered from leaves, 2‐yr‐old trees and winter, respectively. Fungal isolates were initially categorized based on cultural and morphological characters, and representatives of each morphotype were subjected to molecular identifications based on ITS‐rDNA and β‐tubulin sequences. All isolates obtained in this study belonged to the phyla Ascomycota and Basidiomycota assigned to 30 fungal taxa. The most abundant fungal species were Alternaria spp. Species diversity indices of Margalef richness and Shannon–Wiener revealed a high diversity of fungal taxa recovered from leaf and during winter. Frequency (%) of fungal endophytes was highest in winter followed by summer, autumn, and spring significantly, whereas the corresponding sequence was winter, autumn, summer and spring for the diversity of endophytic fungi. Overall, the results of this study based on the community and diversity of endophytic fungal species in C. sinensis showed that the type of plant tissue, season of sampling and age of tree play a driving role on their abundance while their diversity was mainly dependent on the type of plant tissue, season of sampling than on the age of tree.