TAXONOMIC REVISION OF THE FAMILY CASUARINACEAE R. BR., 1814 (ORDER, FAGALES) IN EGYPT

Torreya grandis is known for its medicinal seeds (Wu et al., 2018). Recently, moldy spots were observed on the micropyles of female cones, followed by necrosis of the entire nucellus, resulting in brown rot. In April 2022, the disease incidence reached 68% per cluster (n=132 clusters; 12-14 female cones per cluster) in a production greenhouse. To isolate the pathogen, diseased cones were surface-sterilized in 75% ethanol for 30 s, treated with 2% NaClO for 1 min, rinsed in sterile distilled water, and dried on sterile filter paper. Internal tissues were excised with sterile tweezers, plated on potato dextrose agar (PDA), and incubated at 25 °C in the dark for 7 days. Fungal colonies were purified by repeated subculturing on fresh PDA. Thirteen pure strains were isolated from infected nucellus tissue; 10 of them displayed identical morphology consistent with Cladosporium spp. observed under light microscopy. Colonies were gray-green, round, and velvety with white margins. Conidiophores were apically branched and formed chains of lemon-shaped conidia. Ramoconidia and conidia were cylindrical to globose or obovoid, measuring 4.50-31.40×1.86-5.43 μm and 2.15-9.25×1.06-4.21 μm (n=200), respectively. Based on cultural and morphological characteristics, the fungus was preliminarily identified as Cladosporium tenuissimum (Bensch et al., 2018). To confirm its identity, phylogenetic analysis was conducted using internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF), and actin (ACT) gene regions (Yang et al., 2023). Three representative isolates (TGFC1, TGFC7, and TGFC9) were selected for multi-locus sequencing BLAST analysis showed 99% and 100% identity in ITS sequences with C. tenuissimum (OQ629133 and OR143776), and 100% identity in TEF and ACT sequences with reference strains (TEF: MT154177, OL504967; ACT: MT154174, HM148689). For phylogenetic reconstruction, 15 ex-type Cladosporium strains were included (Bensch et al., 2012). TGFC1, TGFC7, and TGFC9 formed a well-supported clade with C. tenuissimum (CBS 125995). Genetic homogeneity of TGFC1 (GenBank: PQ608378, PQ619083, PQ619082), TGFC7 (GenBank: PV300557, PV346768, PV346769), and TGFC9 (GenBank: PV544812, PV549291, PV549292) was confirmed. For the pathogenicity test, nine healthy female cone clusters of T. grandis "Xifei" were inoculated by spraying a conidial suspension (1×106 conidia/mL) of isolate TGFC1. Nine clusters, sprayed with sterilized water served as controls. The clusters were incubated at 25 °C and 90% relative humidity. After 14 days, the inoculated female cones developed yellow, moldy, and wrinkled symptoms, whereas the control cones remained healthy, showing no signs of fungal growth. C. tenuissimum was successfully reisolated from the diseased cones, thereby fulfilling Koch's postulates. This pathogen causes gray mold and blossom blight in strawberry (Santos et al. 2020). To the best of our knowledge, this is the first report of C. tenuissimum causing moldy spots on the female cones of T. grandis "Xifei". As the disease occurs during the flowering stage, it may impair pollination and seed development, leading to significant economic losses. Therefore, accurate identification of the causal agent is critical for developing effective disease management strategies. Reducing the incidence of female cone disease will improve both yield and quality, contributing to the sustainable development of the Torreya industry.

Five Casuarina species and five Allocasuarina species were inoculated in vitro with three isolates of Pisolithus sp. (Ors.X004 and Ors.7870 from Senegal, PR86 from Australia) to test their ability to form ectomycorrhizas. The mycorrhiza-forming ability varied between fungal isolates. The greatest differences occurred between Casuarina and Allocasuarina species. On Casuarina species, Pisolithus isolates formed only a fungal sheath. However, Ors.X004 induced well-developed ectomycorrhizas on Casuarina equisetifolia, whereas PR86 failed to form any fungal sheath on Casuarina cunninghamiana. On Allocasuarina species, Pisolithus isolates formed generally well-developed ectomycorrhizas. In addition, isolates Ors.7870 and PR86 invaded the cortical cells of Allocasuarina luehmannii and Allocasuarina decaisneana, respectively, thus forming ectendomycorrhizas. Epidermal cells of both Casuarina and Allocasuarina mycorrhizas showed tannin deposits. In fully developed ectomycorrhizas, the epidermal cells were radially elongated and the Hartig net never developed beyond the epidermal cells. In general, the ability to form ectomycorrhizas was more common with the genus Allocasuarina than the genus Casuarina.Key words: Casuarina, Allocasuarina, Pisolithus, ectomycorrhizas.

The presence of hybrids in plant invasions can indicate a potential for rapid adaptation and an added level of complexity in management of the invasion. Three Casuarina tree species, Casuarina glauca, Casuarina cunninghamiana and Casuarina equisetifolia, native to Australia, are naturalized in Florida, USA. Many Florida Casuarina trees are considered unidentifiable, presumably due to interspecific hybridization. We collected tissue from over 500 trees from Australia and Florida and genotyped these using amplified fragment length polymorphisms. Our goal was to determine the exact identity of the Florida species, including any putative hybrid combinations. In Australia, we found high assignment values to the three parental species, and no evidence of hybridization. In Florida, we found many trees with strong assignment to any one of the three species, as well as 49 trees with assignment values intermediate to C. glauca and C. equisetifolia, suggesting hybridization between these species. One population of 10 trees had assignment values intermediate to C. cunninghamiana and C. glauca, suggesting additional hybridization. For 69 of these putative hybrid and parental types, we sequenced a low-copy intron of nuclear G3pdh, and these sequences indicated that some Florida trees contain heterozygotic combinations of C. glauca and C. equisetifolia haplotypes. The presence of novel hybrids in the Florida invasion may enhance evolution of invasive traits in these species. Novel Casuarina hybrids in Florida have no coevolutionary history with any insects or diseases, which may be problematic for biological control efforts.

Casuarina trees are planted along the coast from Hainan province in South China to the Zhoushan Islands of Zhejiang province in Southeastern China. Three key species, Casuarina equisetifolia, Casuarina cunninghamiana and Casuarina glauca, are used as windbreaks, in agroforestry systems, and for the production of timber and fuel wood. Frankia have been studied in China since 1984. Today, Frankia research fields are very wide, and cover morphology, physiology and genetic diversity, and the application of inocula for specific purposes on poor quality sites. In this paper, we review the role of Frankia inoculations in nurseries and casuarina plantations in China and discuss the benefits of inoculation.

A study conducted to determine the ecological status of vegetation under a Casuarina equisetifolia L. plantation revealed that the number of species types, density and biomass of the understorey were drastically reduced compared to an adjoining grassland area. In general, vegetation under Casuarina was characterised by the presence of a few dominants with a trend towards homogeneity. On the other hand, the vegetation in the grassland was heterogeneous, with conspicuous spatial pattern and, thus, more stable. Indices of richness (indicating numerical strength), evenness (representing spatial distribution), and diversity (combining both richness and evenness) also indicated a marked difference in the vegetation between the two sites. The Shannon index, indices of richness and evenness, and Hill. s diversity measures were greater in grassland areas than in Casuarina plantation, thereby signifying a richer, more diverse and even vegetation in the grassland. In contrast, the Simpson index of dominance which shows an inverse relation with diversity, was greater under the Casuarina plantation. The reasons for the restrained vegetation under Casuarina were explored in terms of allelopathic interference of various tree parts (fresh as well as fallen) as one of the major factor. The leaf leachates collected under the canopy of Casuarina trees in the plantation were found to have deleterious effect on the growth of Medicago sativa and Ageratum conyzoides and were rich in phenolics. The extracts prepared from different tree parts such as needles, female cones and litter adversely affected the growth and dry weight accumulation in M. sativa and A. conyzoides. An appreciable amount of water-soluble phenolics, known phytotoxins, was estimated in the fog leachates and different plant parts under use. It is concluded that phenolics released from the green needles and litter of the tree adversely affect the understorey vegetation.

Casuarina (Casuarina equisetifolia L.) is a species used like wind break, in streets ornamentation, soils conservation and dunes fixation. The main form of propagation of this species is by seeds, although, in nursery, the fungus Fusarium lacertarum have been cause damping-off in seedlings. Thus, the objective of this work was to characterize morphologically, physiologically and molecularly the pathogen. To molecular characterization, pure colonies were cultivated in PDA and LCA culture media, and for physiological characterization, colonies were cultivated in PDA, SNA, Mathur and V8 culture media. The genomic segment most appropriated to identify this species was the tef1-?. The causal agent of damping-off in Casuarina equisetifolia seedlings was confirmed like F. lacertarum, and the most efficient genomic region to identification is tef1-?. The best incubation condition to conidia production of F. lacertarum was in Mathur medium and the largest daily average growth was in SNA medium. The morphological characteristics are equivalent to the characterization of F. incarnatum-equiseti species complex isolated.

Araucaria angustifolia (Bert.) O. Kuntze is a dioecious conifer species native of Brazil. The rare occurrence of monoiceous specimens have been attributed to pathogenic infections or other injuries in adult trees. Here, the morphological characteristics of male and female cones and pollen grains of a monoiceous A. angustifolia are described. Male and female cones and pollen grains presented normal morphology, lacking any sort of injuries or infection and suggesting the existence of further grounds for the occurrence of monoicy in this conifer species.

The ironwood tree (Casuarina equisetifolia, family Casuarinaceae), an indigenous agroforestry species in Guam, has been threatened by ironwood tree decline (IWTD) since 2002. Formation of bacterial ooze by the wilt pathogen from the Ralstonia solanacearum species complex and wetwood bacteria (primarily Klebsiella species) has been linked to IWTD. In addition, termite infestation of trees was statistically associated with IWTD. Termites are known carriers of a diverse microbiome. Therefore, we hypothesized that termites could be vectors of bacteria linked to IWTD. To investigate the potential role of termites as pathogen vectors, we employed next-generation 16S rRNA gene sequencing to describe the bacteria diversity of Nasutitermes takasagoensis (Family Termitidae) workers collected from 42 ironwood trees of different disease stages in Guam in association with tree-, plot-, and location-related factors. Nasutitermes takasagoensis workers account for the majority of termite infestations of ironwood trees. The bacterial phyla composition of N. takasagoensis workers was typical for wood-feeding higher termites consisting mainly of Spirochaetes and Fibrobacteres. However, Ralstonia species were not detected and Klebsiella species were rare even in termites collected from trees infected with Ralstonia and wetwood bacteria. Feeding experiments suggested that termites prefer to consume wood with low pathogen content over wood with high pathogen load. Termites were able to ingest Ralstonia but Ralstonia could not establish itself in healthy termite bodies. We concluded that N. takasagoensis workers are not vectors for Ralstonia spp. or the bacterial endophytes associated with wetwood (Klebsiella, Pantoea, Enterobacter, Citrobacter, and Erwinia) that were previously observed in IWTD-infested trees. The bacterial diversity in termite samples was significantly influenced by various factors, including Tree Health, Site Management, Plot Average Decline Severity, Proportion of Dead Trees in the Plot, Proportion of Trees with Termite Damage in the Plot, Presence of Ralstonia, and Altitude.

The ironwood tree (Casuarina equisetifolia, family Casuarinaceae), an indigenous agroforestry species in Guam, has been threatened by ironwood tree decline (IWTD) since 2002. Formation of bacterial ooze by the wilt pathogen from the Ralstonia solanacearum species complex and wetwood bacteria (primarily Klebsiella species) has been linked to IWTD. In addition, termite infestation of trees was statistically associated with IWTD. Termites are known carriers of a diverse microbiome. Therefore, we hypothesized that termites could be vectors of bacteria linked to IWTD. To investigate the potential role of termites as pathogen vectors, we employed next-generation 16S rRNA gene sequencing to describe the bacteria diversity of Nasutitermes takasagoensis (Family Termitidae) workers collected from 42 ironwood trees of different disease stages in Guam in association with tree-, plot-, and location-related factors. Nasutitermes takasagoensis workers account for the majority of termite infestations of ironwood trees. The bacterial phyla composition of N. takasagoensis workers was typical for wood-feeding higher termites consisting mainly of Spirochaetes and Fibrobacteres. However, Ralstonia species were not detected and Klebsiella species were rare even in termites collected from trees infected with Ralstonia and wetwood bacteria. Feeding experiments suggested that termites prefer to consume wood with low pathogen content over wood with high pathogen load. Termites were able to ingest Ralstonia but Ralstonia could not establish itself in healthy termite bodies. We concluded that N. takasagoensis workers are not vectors for Ralstonia spp. or the bacterial endophytes associated with wetwood (Klebsiella, Pantoea, Enterobacter, Citrobacter, and Erwinia) that were previously observed in IWTD-infested trees. The bacterial diversity in termite samples was significantly influenced by various factors, including Tree Health, Site Management, Plot Average Decline Severity, Proportion of Dead Trees in the Plot, Proportion of Trees with Termite Damage in the Plot, Presence of Ralstonia, and Altitude.

DNA extracted directly from nodules was used to assess the genetic diversity of Frankia strains symbiotically associated with two species of the genus Casuarina and two of the genus Allocasuarina naturally occurring in northeastern Australia. DNA from field-collected nodules or extracted from reference cultures of Casuarina-infective Frankia strains was used as the template in PCRs with primers targeting two DNA regions, one in the ribosomal operon and the other in the nif operon. PCR products were then analyzed by using a set of restriction endonucleases. Five distinct genetic groups were recognized on the basis of these restriction patterns. These groups were consistently associated with the host species from which the nodules originated. All isolated reference strains had similar patterns and were assigned to group 1 along with six of the eight unisolated Frankia strains from Casuarina equisetifolia in Australia. Group 2 consisted of two unisolated Frankia strains from C. equisetifolia, whereas groups 3 to 5 comprised all unisolated strains from Casuarina cunninghamiana, Allocasuarina torulosa, and Allocasuarina littoralis, respectively. These results demonstrate that, contrary to the results of previous molecular studies of isolated strains, there is genetic diversity among Frankia strains that infect members of the family Casuarinacaeae. The apparent high homogeneity of Frankia strains in these previous studies probably relates to the single host species from which the strains were obtained and the origin of these strains from areas outside the natural geographic range of members of the family Casuarinaceae, where genetic diversity could be lower than in Australia.

The utility of isozyme analysis in elucidating the relationship between Australian tree taxa is reviewed. Although little exploited to date, isozyme analysis is shown to be a rapid and relatively powerful method of examining relationships, if used at an appropriate taxonomic level. For Eucalyptus and Acacia, isozymes appear to be the most informative at the lower taxonomic levels. In Eucalyptus delegatensis and Casuarina cunninghamiana, isozyme data strongly support subspecies erected on the basis of morphological characteristics. In Acacia holoserocea, isozyme data predicted the existence of two subspecies, which prediction as later supported by morphological characters. An isozyme study of the phylogenetic relationships within the 'green ash' group of eucalypts yielded a phylogenetic hypothesi comparable to one derived from morphological characters, but also highlighted areas of discrepancy requiring further research. At the generic level, isozyme data for the Australian species of Litsea and Neolitsea successfully separated the two genera and allowed phylogenetic relationships within genera to be hypothesised. For the larger tree genera such as the Eucalyptus and Acacia, however, the utility of isozymes at the higher taxonomic levels is likely to be low, because of the difficulty in establishing homologies between taxa, or insufficient phylogenetic information when taxa being compared have few alleles in common.

The newly recognized extant Asian conifer genus Cathaya is represented by fossil material in Europe. Female cones from the Oligocene of Saxony are assigned to C. europaea n. sp. ; these are distinguishable from the cones of the extant species of Cathaya. Cathaya abchasica n. sp. is based on foliar remains from the Pliocene of Abkhazia; the generic assignment is validated by comparison of the stomatal characteristics. The one previous fossil record of Cathaya in Europe is not considered valid. — J. A. Wolfe

Of the six juniper species found in the Bulgarian flora, three of the species have controversial taxonomic positions. Juniperus pygmaea K. Koch and J. sibirica Burgsd. exhibit similar morphological characteristics to J. communis L. in terms of leaves and female cones (galbuli). This is one of the reasons why, in the recent taxonomic developments, J. pygmaea and J. sibirica were united in a common variety of J. communis, namely, J. communis var. saxatilis. However, such a grouping of species in the Flora of Bulgaria has not been adopted. This study aimed to evaluate the degrees of similarity or difference in the structure of the leaves, galbuli, seeds, and pollen of J. communis, J. sibirica, and J. pygmaea using the methods of comparative anatomy by light microscope (LM) and scanning electron microscopy (SEM) observations and complex morphological measurements. The working hypothesis of this study was that the three species would show a different degree of similarity with each other, which would clarify their taxonomic rank. The morphological parameters revealed differences between the length/width ratio of galbuli and seed length of the three species, while leaf characteristics (length and width) showed a stronger resemblance between J. sibirica and J. pygmaea. Furthermore, a greater distinction between the leaves and galbili of J. communis and J. sibirica was found. The SEM analyses showed variations in the seed shape and spermoderm among the three species. The shape of J. communis seeds was oval and elongated, while J. pygmaea seeds were pear-shaped, and J. sibirica seeds were triangular-rhombic. The length and height of striations were diverse on seed spermoderm in the three species. The epicuticular waxes of leaves, located on the tips of the anticlinal walls of the elongated epidermal cells in J. pygmaea and J. communis, were oval, while they formed raised comb-like crystals in J. sibirica. The morphological, anatomical, and SEM analysis affirmed the accepted taxonomic status of J. communis and J. sibirica as independent species within the Bulgarian flora. Based on most of the analyzed parameters, J. pygmaea exhibits significant similarity with J. sibirica. Additionally, the similar habitats of these two species support the determination of J. pygmaea as a variety or form of J. sibirica rather than J. communis (J. sibirica forma pygmaea).

Osteoporosis is a frequent skeletal disease characterised both by loss of bone mineral mass and deterioration of cancellous bone micro-architecture. It can be caused by mechanical disuse, estrogen deficiency or natural age-related resorption process. Numerical analysis of high-resolution images of the trabecular network is recognised as a powerful tool for assessment of structural characteristics. Using μCT images of 73 thoracic and 78 lumbar human vertebral specimens in vitro with isotropic resolution of 26μm we simulate bone atrophy as random resorption of bone surface voxels. Global morphological and topological characteristics provided by four Minkowski Functionals (MF) are calculated for two numerical resorption models with and without conservation of global topological connectivity of the trabecular network, which simulates different types of bone loss in osteoporosis, as it has been described in males and females. Diagnostic performance of morphological and topological characteristics as a function of relative bone loss is evaluated by a correlation analysis with respect to experimentally measured Maximum Compressive Strength (MCS). In both resorption models the second MF, which coincides with bone surface fraction BS/TV, demonstrates almost constant value of Pearson's correlation coefficient with respect to the relative bone loss ▵BV/TV. This morphological characteristic does not vary considerably under age-related random resorption and can be used for predicting bone strength in the elderly. The third and fourth MF demonstrate an increasing correlation coefficients with MCS after applying random bone surface thinning without preserving topological connectivity, what can be used for improvement of evaluation of the current state of the structure.

The Genus Casuarina is a coastal plant distributed throughout South East Asia, Australia, and the Pacific islands. In Taiwan, more than 10 species of Casuarina have been introduced based on the proposition that these drought tolerant, fast growing trees would be suitable for windbreaks and would help stabilize coastal sand. The genetic diversity of three Casuarina species grown in Taiwan, Casuarina equisetifolia, C.glauca, and C. cunninghamiana was determined and compared to those of native populations grown in Australasia as part of an international provenance test using the Inter-Simple Sequence Repeat (ISSR) marker system. Based on our results, the average Nei gene diversity of Casuarina grown in Taiwan (0.1940) is considerably higher (p < 0.001) than that of native populations of C. equisetifolia (0.1288), C. cunninghamiana (0.0922), and C. glauca (0.0577). Cluster and principal component analyses indicated that the Taiwan-grown trees are more closely related to C. equisetifolia than either C. cunninghamiana or C. glauca. By integrated reasoning of genetic variation and branchlet morphology, we may conclude that coastal Casuarina plants currently survived and grown in Taiwan are the products of introgressive hybridization involving C. equisetifolia, C. glauca, and possibly C. cunninghamiana. Key words: Casuarina, genetic diversity, hybridization, inter simple sequence repeat (ISSR).