Detection of phytoplasma infections in declining Populus nigra ‘Italica’ trees and molecular differentiation of the aster yellows phytoplasmas identified in various Populus species

A disease of Populus nigra‘Italica’ associated with foliar yellowing, sparse foliage, stunting, dieback, and decline was observed in south-western Germany; a witches’ broom disease of Populus alba that is known in other countries was also detected in Hungary and Germany. The aetiology of the diseases was studied by fluorescence microscopy and polymerase chain reaction (PCR) amplification. Using fluorescence microscopy, phytoplasmas could be detected only in P. alba. However, most diseased trees of P. nigra‘Italica’ tested phytoplasma-positive by PCR. In some of the trees the phytoplasma numbers were so low that nested PCR was required to detect the infection. Very low phytoplasma numbers were also observed in diseased Populus tremula. The identity of phytoplasmas from P. nigra‘Italica’ sampled in Germany and France, P. alba and also P. tremula was examined by restriction fragment length polymorphism (RFLP) analysis of PCR-amplified ribosomal DNA. In all poplars, phytoplasmas of the aster yellows group were detected. However, three different RFLP groups were identified that consisted of (1) French strains from P. nigra‘Italica’, (2) German strains from P. nigra‘Italica’ and (3) strains from P. alba and P. tremula. The profile observed in the last group was probably the result of sequence heterogeneity in the two 16S RNA genes.     

Identification of the Armillaria root rot pathogen in Ethiopian plantations

Armillaria root rot is a well‐known disease on a wide range of plants, world‐wide. In Ethiopia, the disease has previously been reported on Pinus spp., Coffea arabica and on various native hardwoods. The causal agent of the disease has been attributed to Armillaria mellea, a species now known to represent a complex of many different taxa. The aim of this study was to determine the extent of Armillaria root rot and the identity of the Armillaria sp. in Ethiopian plantations. As part of a plantation disease survey in 2000 and 2001, samples were collected in plantations at and around Munessa Shashemene, Wondo Genet, Jima, Mizan and Bedele, in south and south‐western Ethiopia. Basidiocarps were collected and their morphology studied. Morphological identification was confirmed by sequencing the intergenic spacer (IGS‐1) region of the ribosomal rRNA operon and comparing data with published sequences of Armillaria spp. Armillaria isolates were collected from Acacia abyssinica, Pinus patula, Cedrela odorata and Cordia alliodora trees. Sporocarps were found on stumps of native Juniperus excelsa. Basidiocarp morphology and sequence data suggested that the fungus in Ethiopia is similar to that causing disease of Pinus spp. in South Africa and previously identified as A. fuscipes. This identification was confirmed for all isolates, based on sequence data. Armillaria fuscipes is known to be common in southern Africa. Its widespread occurrence in Ethiopia suggests that it is also the major cause of Armillaria root rot in that country.     

Micropropagation and tissue culture of Eucalyptus-a review

Micropropagation has the potential to provide very high multiplication rates of selected tree genotypes, with resulting short-term silvicultural gains. Aseptic cultures have been established from seeds, seedlings, shoots, flowers and lignotubers. Callus cultures have been established from a wide range of tissue sources for at least 30 species of Eucalyptus. Plant regeneration from callus was successful for 12 of these species. Micropropagation through axillary proliferation, or adventitious shoot proliferation on nodal explants, or both, has been successful. An agar-based medium of Murashige and Skoog with a low auxin/cytokinin ratio is most commonly used for shoot multiplication. Vitrification and shoot senescence remain problems. Gibberellic acid was added in some media to stimulate shoot elongation. Various media are used for in vitro root initiation. Suspension and protoplast cultures have been achieved and plants have been regenerated from protoplasts. In vitro techniques are presently being applied to Eucalyptus to achieve genetic transformations.     

Testing of selected South African <Emphasis Type="Italic">Pinus</Emphasis> hybrids and families for tolerance to the pitch canker pathogen, <Emphasis Type="Italic">Fusarium circinatum</Emphasis>

Plantations of Pinus spp. constitute approximately 50% of the South African forestry industry. The first aim of this study was to develop a reliable inoculation technique to screen Pinus spp., for tolerance to infection by F. circinatum, which threatens pine forestry in South Africa. Inoculation of branches was compared with stem inoculations and we considered the number of branches or trees required to obtain statistically significant results. Furthermore, variation in the susceptibility of some Pinus families, clones and hybrids was considered. Results showed that branch inoculations were closely correlated with those from stem inoculations, and that it is important to consider branch and stem diameters when assessing susceptibility of trees. Subsequent trials using branch inoculations showed significant differences in F. circinatum tolerance amongst a range of pine species and hybrids of potential interest to forestry in South Africa. Significant differences in susceptibility were also found among clones of two P. radiata families. The most tolerant trees were P. elliottii × caribaea and P. patula × oocarpa hybrids, while the most susceptible species were P. patula, P. greggii and hybrids of these two. This is the first trial considering the susceptibility of Pinus hybrids, Pinus clones and some P. patula provenances, and the results indicate excellent potential for breeding for tolerance to pitch canker in South Africa. Application The accurate selection of disease tolerant planting stock for the South African forestry industry is crucially important for the continued sustainability of this important industry. The work described here provides valuable information on an artificial inoculation technique that will assist the industry in screening trees for tolerance to the pitch canker fungus, F. circinatum. It also provides some indication of the relative susceptibility of a number of Pinus spp., hybrids and families currently being evaluated in the country.     

Mycosphaerella species associated with leaf disease of Eucalyptus globulus in Ethiopia

Eucalyptus spp. are among the most widely planted exotic trees in Ethiopia. Several damaging leaf pathogens are known from Eucalyptus spp. worldwide. Of these, Mycosphaerella spp. are among the most important, causing the disease known as Mycosphaerella leaf disease (MLD). Characteristic symptoms of MLD include leaf spot, premature defoliation, shoot and twig dieback. Recent disease surveys conducted in Ethiopian Eucalyptus plantations have revealed disease symptoms similar to those caused by Mycosphaerella spp. These symptoms were restricted to E. globulus trees growing in several localities in south, south western and western Ethiopia. The aim of this study was to identify the fungi associated with this disease. This was achieved by examining ascospore germination patterns, anamorph associations and sequence data from the Internal Transcribed Spacer (ITS) region of the rRNA operon, for representative isolates. Several different ascospore germination patterns were observed, suggesting that more than one species of Mycosphaerella is responsible for MLD on E. globulus in Ethiopia. Analysis of sequence data showed that three Mycosphaerella spp., M. marksii, M. nubilosa and M. parva were present. This is the first report of these three species from Ethiopia and represents a valuable basis on which to build further studies in the region.     

Characterization of Botryosphaeriaceae from plantation‐grown Eucalyptus species in South China

The Botryosphaeriaceae is a species‐rich family that includes pathogens of a wide variety of trees, including Eucalyptus species. Symptoms typical of infection by the Botryosphaeriaceae have recently been observed in Eucalyptus plantations in South China. The aim of this study was to identify the Botryosphaeriaceae associated with these symptoms. Isolates were collected from branch cankers and senescent twigs of different Eucalyptus spp. All isolates resembling Botryosphaeriaceae were separated into groups based on conidial morphology. Initial identifications were made using PCR‐RFLP fingerprinting, by digesting the ITS region of the rDNA operon with the restriction enzymes CfoI and KspI. Furthermore, to distinguish isolates in the Neofusicoccum parvum/N. ribis complex, a locus (BotF15) previously shown to define these species, was amplified and restricted with CfoI. Selected isolates were then identified using comparisons of DNA sequence data for the ITS rDNA and translation elongation factor 1‐alpha (TEF‐1α) gene regions. Based on anamorph morphology and DNA sequence comparisons, five species were identified: Lasiodiplodia pseudotheobromae, L. theobromae, Neofusicoccum parvum, N. ribis sensu lato and one undescribed taxon, for which the name Fusicoccum fabicercianum sp. nov. is provided. Isolates of all species gave rise to lesions on the stems of an E. grandis clone in a glasshouse inoculation trial and on the stems of five Eucalyptus genotypes inoculated in the field, where L. pseudotheobromae and L. theobromae were most pathogenic. The five Eucalyptus genotypes differed in their susceptibility to the Botryosphaeriaceae species suggesting that breeding and selection offers opportunity for disease avoidance in the future.     

Conservation planning with spatially explicit models: a case for horseshoe bats in complex mountain landscapes

Context

Context Bats are considered as an ecological indicator of habitat quality due to their sensitivity to human-induced ecosystem changes. Hence, we will focus the study on two indicator species of bats as a proxy to evaluate structure and composition of the landscape to analyze anthropic pressures driving changes in patterns.

Objectives

This study develops a spatially-explicit model to highlight key habitat nodes and corridors which are integral for maintaining functional landscape connectivity for bat movement. We focus on a complex mountain landscape and two bat species: greater (Rhinolophus ferrumequinum) and lesser (Rhinolophus hipposideros) horseshoe bats which are known to be sensitive to landscape composition and configuration.

Methods

Species distribution models are used to delineate high-quality foraging habitat for each species using opportunistic ultrasonic bat data. We then performed connectivity analysis combining (modelled) suitable foraging habitat and (known) roost sites. We use graph-theory and the deviation in the probability of connectivity to quantify resilience of the landscape connectivity to perturbations.

Results

Both species were confined to lowlands (<1000 m elevation) and avoided areas with high road densities. Greater horseshoe bats were more generalist than lesser horseshoe bats which tended to be associated with broadleaved and mixed forests.

Conclusions

The spatially-explicit models obtained were proven crucial for prioritizing foraging habitats, roost sites and key corridors for conservation. Hence, our results are being used by key stakeholders to help integrate conservation measures into forest management and conservation planning at the regional level. The approach used can be integrated into conservation initiatives elsewhere.

     

The pathogenic potential of endophytic Botryosphaeriaceous fungi on Terminalia species in Cameroon

In Cameroon, native Terminalia spp. represent an important component of the forestry industry, but limited information is available regarding the fungal pathogens that affect them. The Botryosphaeriaceae are endophytic fungi and latent pathogens that can result in wood stain, cankers, die‐back and death of trees, particularly when trees are under stress. The aim of this study was, therefore, to identify and characterize the Botryosphaeriaceae occurring as endophytes of Terminalia spp. in Cameroon, as part of a larger project to identify potential pathogens of these trees in the country. Samples were collected from three Terminalia spp. in the Central, Southern and Eastern Regions and the resultant Botryosphaeriaceae were identified using morphology and DNA sequence comparisons for the ITS and tef 1‐α gene regions. Furthermore, inoculation trials were conducted to consider the relative pathogenicity of the isolates collected. The majority of isolates (88%) represented species of Lasiodiplodia, including L. pseudotheobromae, L. theobromae and L. parva. The remaining isolates were identified as Endomelanconiopsis endophytica. Pathogenicity trials on young T. mantaly and T. catappa trees revealed that L. pseudotheobromae was the most pathogenic species followed by L. theobromae.     

Molecular tracing of <Emphasis Type="Italic">Bradyrhizobium </Emphasis>strains helps to correctly interpret <Emphasis Type="Italic">Acacia mangium </Emphasis>response to inoculation in a reforestation experiment in Madagascar

A field inoculation experiment using two Bradyrhizobium sp. strains was set up in Madagascar to test the growth response of Acacia mangium and to follow up the survival of inoculant strains using molecular tools. Three months after inoculation, one of the inoculant strains, AUST13c, exhibited a marked growth-promoting effect with a shoot height about 40% higher than that of the uninoculated control plants or TEL8-inoculated plants. The positive effect on tree growth initially observed with AUST13c was no more significant 6 months after transfer to the field and disappeared completely at 13 and 19 months. Analyses of nodule bacterial rRNA by PCR/RFLP displayed an early contamination of the different inoculation treatments by AUST13c 3 months after inoculation, spreading to almost all nodules of the trial 6 months later. This work clearly demonstrated that the progressive reduction of the positive effect of AUST13c inoculation on tree growth after field transplanting was not due to a progressive disappearance of this introduced strain but, on the contrary, was related to the widening spreading of AUST13c in all the plots. This was attributed to a higher competitiveness and effectiveness of AUST13c over the local strains and TEL8.     

Detection and stability of the major almond allergen in foods

Almond major protein (AMP or amandin), the primary storage protein in almonds, is the major allergen recognized by almond-allergic patients. A rabbit antibody-based inhibition ELISA assay for detecting and quantifying AMP in commercial foods has been developed, and this assay, in conjunction with Western blotting analyses, has been applied to the investigation of the antigenic stability of AMP to harsh food-processing conditions. The ELISA assay detects purified AMP at levels as low as 87 +/-16 ng/mL and can detect almond at between 5 and 37 ppm in the tested foods. The assay was used to quantify AMP in aqueous extracts of various foods that were defatted and spiked with known amounts of purified AMP or almond flour. In addition, AMP was quantified in commercially prepared and processed almond-containing foods. Neither blanching, roasting, nor autoclaving of almonds markedly decreased the detectability of AMP in subsequent aqueous extracts of almonds. Western blots using both rabbit antisera and sera from human almond-allergic patients confirm a general stability of the various peptides that comprise this complex molecule and show that the rabbit antibody-based assay recognizes substantially the same set of peptides as does the IgE in sera from almond-allergic patients.