Over-expression of a cacao class I chitinase gene in Theobroma cacao L. enhances resistance against the pathogen, Colletotrichum gloeosporioides

Theobroma cacao L. plants over-expressing a cacao class I chitinase gene (TcChi1) under the control of a modified CaMV-35S promoter were obtained by Agrobacterium-mediated transformation of somatic embryo cotyledons. Southern blot analysis confirmed insertion of the transgene in eight independent lines. High levels of TcChi1 transgene expression in the transgenic lines were confirmed by northern blot analysis. Chitinase activity levels were measured using an in vitro fluorometric assay. The transgene was expressed at varying levels in the different transgenic lines with up to a sixfold increase of endochitinase activity compared to non-transgenic and transgenic control plants. The in vivo antifungal activity of the transgene against the foliar pathogen Colletotrichum gloeosporioides was evaluated using a cacao leaf disk bioassay. The assay demonstrated that the TcChi1 transgenic cacao leaves significantly inhibited the growth of the fungus and the development of leaf necrosis compared to controls when leaves were wound inoculated with 5,000 spores. These results demonstrate for the first time the utility of the cacao transformation system as a tool for gene functional analysis and the potential utility of the cacao chitinase gene for increasing fungal pathogen resistance in cacao.     

Bacillus subtilis and Enterobacter cloacae endophytes from healthy Theobroma cacao L. trees can systemically colonize seedlings and promote growth

Clonal genotypes resistant to fungal diseases are an important component of the cocoa production system in southeastern Bahia state (Brazil), so that technologies for faster production of stronger and healthier plantlets are highly desirable. In this study, the effects of inoculated bacterial endophytes isolated from healthy adult cacao plants on seedlings, and aspects related to inoculation methods, colonization patterns, and photosynthesis were investigated. Sequencing of 16S rRNA, hsp-60, and rpo-B genes placed the wild-type isolates within the species Enterobacter cloacae (isolates 341 and 344) and Bacillus subtilis (isolate 629). Spontaneous rifampicin-resistant (rif^R) variants for 344 were also produced and tested. Endophytic application was either by immersion of surface sterilized seeds in bacterial suspensions or direct inoculation into soil, 20 days after planting non-inoculated seeds into pots. Results from in vitro recovery of inoculated isolates showed that the wild-type endophytes and rif^R variants systemically colonized the entire cacao seedlings in 15–20 days, regardless of the inoculation method. Some endophytic treatments showed significant increases in seedlings’ height, number of leaves, and dry matter. Inoculation methods affected the combined application of endophytes, which maintained the growth-promotion effects, but not in the same manner as in single applications. Interestingly, the 344-3.2 rif^R variant showed improved performance in relation to both the wild type and another related variant. Photosynthetic rates and stomatal conductance increased significantly for some endophytic treatments, being partially associated with effects on growth and affected by the inoculation method. The results suggest that E. cloacae and B. subtilis endophytes from healthy adult plants (not transmitted by seeds) were able to promote vegetative growth on cacao seedlings. The development of products for large-scale use in seedlings/plantlets production systems was discussed.     

PCR-based detection and characterization of the fungal pathogens Colletotrichum gloeosporioides and Colletotrichum capsici causing anthracnose in papaya (Carica papaya l.) in the Yucatan peninsula

Colletotrichum gloeosporioides is the common causal agent of anthracnose in papaya (Carica papaya L.) fruits, and infection by this fungal pathogen results in severe post-harvest losses. In the Yucatán peninsula (Mexico) a different Colletotrichum species was isolated from papaya fruits with atypical anthracnose lesions. The DNAs from a variety of Colletotrichum isolates producing typical and atypical lesions, respectively, were amplified by PCR with C.gloeosporioides-specific primers. All isolates from typical anthracnose lesions yielded a 450 bp PCR product, but DNAs from isolates with atypical lesions failed to produce an amplification product. For further characterization, the rDNA 5.8S-ITS region was amplified by PCR and processed for sequencing and RFLP analysis, respectively, to verify the identity of the papaya anthracnose pathogens. The results revealed unequivocally the existence of two Colletotrichum species causing anthracnose lesions on papaya fruits: C. gloeosporioides and C. capsici. PCR-RFLP using the restriction endonuclease MspI reliably reproduced restriction patterns specific for C. capsici or C. gloeosporioides. The generation of RFLP patterns by MspI (or AluI or RsaI) is a rapid, accurate, and unequivocal method for the detection and differentiation of these two Colletotrichum species.     

Differentiation of lineages within “Colletotrichum gloeosporioides s.l.” associated with cassava anthracnose disease by BOX‐ and ERIC‐PCRs

Several molecular techniques have been used to differentiate species or genetic lineages of microorganisms prior to sequencing. Among them, BOX‐ and ERIC‐PCRs may provide specific banding patterns for different species, allowing its differentiation. Therefore, the objective of this study was to evaluate these techniques as a tool for differentiation of phylogenetic lineages belonging to the Colletotrichum gloeosporioides species complex associated with cassava anthracnose disease. Sets of BOX‐ and ERIC‐PCR primers were used to assess the differentiation of lineages belonging to the complex with 81 C. gloeosporioides sensu lato (s.l.) isolates from different cassava producing regions. Some were identified by sequencing, such as Colletotrichum fructicola, Colletotrichum tropicale, C. gloeosporioides s.s, Colletotrichum theobromicola, Colletotrichum siamense, Colletotrichum brevisporum and Colletotrichum sichuanensis. The primers were able to amplify DNA fragments from all isolates. The ERIC‐PCR presented a wider range of banding patterns in comparison to BOX‐PCR, providing better differentiation of the individuals, as well as a higher correlation with the phylogenetic data was obtained by ERIC‐PCR and the combined data set for “BOX‐/ERIC‐PCRs,” inferred by Mantel test. However, the use of concatenated data (BOX‐/ERIC‐PCRs) reduced the discriminatory capacity presented by ERIC‐PCR alone, probably due to the lowest resolution of BOX‐PCR. Therefore, ERIC‐PCR technique enabled efficient differentiation of isolates belonging to the C. gloeosporioides complex and can be used to analyse multiple isolates in a collection and also being an important tool as a guide in the decision‐making process prior to sequencing. Based on this methodology, it was possible to identify two new species associated with cassava anthracnose disease, C. brevisporum and C. sichuanensis, being the first report of these two species associated with cassava anthracnose disease in Brazil.     

Canopy cover and leaf age affect colonization by tropical fungal endophytes: Ecological pattern and process in Theobroma cacao (Malvaceae)

Fungal endophytes inhabit healthy tissues of all terrestrial plant taxa studied to date and are diverse and abundant in leaves of tropical woody angiosperms. Studies have demonstrated that plant location and leaf age influence density of endophyte infection in leaves of tropical forest trees. However, ecological factors underlying these observations have not been explored in detail. Here, we establish that foliar endophytes of a tropical tree (Theobroma cacao, Malvaceae) are transmitted horizontally and that endophyte-free seedlings can be produced for experimental manipulation by protecting aerial tissues from surface wetting. At Barro Colorado Island, Panama, we used transects of endophyte-free seedlings to determine the importance of several factors (canopy cover, abundance of aerial and epiphytic propagules, leaf age, leaf chemistry, leaf toughness and duration of exposure to viable air spora) in shaping colonization by endophytic fungi. Endophytes colonized leaves of T. cacao more rapidly beneath the forest canopy than in cleared sites, reflecting local abundance of aerial and epiphytic propagules. The duration of exposure, rather than absolute leaf age, influenced endophyte infection, whereas leaf toughness and chemistry had no observed effect. Endophytes isolated from mature T. cacao grew more rapidly on media containing leaf extracts of T. cacao than on media containing extracts from other co-occurring tree species, suggesting that interspecific differences in leaf chemistry influence endophyte assemblages. Together, these data allow us to identify factors underlying patterns of endophyte colonization within healthy leaves of this tropical tree.     

Characterization of Colletotrichum gloeosporioides isolates from avocado and almond fruits with molecular and pathogenicity tests.

One hundred twenty isolates of Colletotrichum gloeosporioides from avocado (6 U.S. and 57 Israeli isolates) and almond (57 Israeli isolates) fruits were compared by various molecular methods and a pathogenicity assay in order to determine the genetic diversity and host specificity between and among the different populations. DNA from eight additional U.S. almond anthracnose isolates were also compared. PCR amplification of genomic DNA with four primers produced uniform banding patterns for all the Israeli almond isolates from different geographic locations in Israel. DNAs from the U.S. almond isolates were distinct from DNAs of the Israeli isolates. In contrast, the avocado isolates from Israel and the United States were more diverse, with numerous arbitrarily primed-PCR phenotypes being observed. HaeIII digestion patterns of A+T-rich DNA distinguished between the almond and avocado isolates. Southern hybridization of the repetitive nuclear-DNA element GcpR1 to PstI-digested genomic DNA of almond and avocado isolates revealed no polymorphic fragments among the almond isolates, whereas polymorphic fragments were observed among the avocado isolates. Amplification and subsequent restriction enzyme digestion of the internal transcribed spacer 4 and 5 regions between the small and large nuclear subunits of DNA encoding rRNA failed to distinguish between C. gloeosporioides isolates from a diverse host range. In artificial inoculations, avocado isolates produced various lesions on avocado and almond fruits, whereas the almond isolates infected both fruits at a lower rate.     

Diversity of fungal latent pathogens and true endophytes associated with fruit trees in Uruguay

We have explored the fungal diversity in asymptomatic twigs of apple, peach, pear and blueberry trees, with the objective of discerning between true endophytes and latent pathogens. Several fungal genera containing known bark pathogens were found. Seven Diaporthe species—D. oxe, D. infecunda, D. serafiniae, D. phaseolorum, D. terebinthifolii, D. foeniculina and D. brasiliensis—were identified, along with Botryosphaeria dothidea, Neofusicoccum parvum, Neofusicoccum australe, Cytospora sp., Cytospora acaciae and Pestalotiopsis spp. A pathogenicity trial was undertaken to determine the role of these species on apple, pear, blueberry and peach shoots. Diaporthe brasiliensis, D. foeniculina, Diaporthe inconspicua, D. terebinthifolii, Diaporthe sp.1, Cytospora‐like isolates and Pestalotiopsis spp. isolates produced no lesions on inoculated shoots, suggesting that they could be considered true endophytes on their respective hosts. Meanwhile, some of the isolates of Diaporthe—D. oxe, Diaporthe sp.2, D. infecunda and D. serafiniae, B. dothidea, N. parvum and N. australe could be regarded as latent pathogens in their respective hosts as they produced sunken cankers and necrosis on inoculated shoots. These results demonstrate that apple, pear, blueberry and peach healthy shoots can host many known endophytic fungi along with potential wood disease‐causing fungi that should be regarded as latent pathogens.     

Trypanosoma rangeli sialidase: lack of activity in stocks from Panama and other regions

A total of 33 crude and cloned Trypanosoma rangeli stocks found as natural infections in human from Panama and other endemic areas of Central and South America were evaluated as producers of sialidase (SA) activity through the MU-NANA fluorescence test. Negative results were observed in 6 of the isolates: Panama (4), Honduras (1), and Brazil (1). In addition, an immunoblotting analysis confirm the presence of the SA antigen in these stocks without enzymatic activity. These findings must be considered in the interpretation of the biological significance of T. rangeli SA and in the proper characterization and identification of this parasite.     

Ecology of bacterial endophytes associated with wetland plants growing in textile effluent for pollutant-degradation and plant growth-promotion potentials

In this study, 41 culturable endophytic bacteria were isolated from the roots and shoots of three wetland plants, Typha domingensis, Pistia stratiotes and Eichhornia crassipes, and identified by 16S rRNA gene sequencing. Textile effluent-degrading and plant growth-promoting activities of these endophytes were determined. The analysis of endophytic bacterial communities indicated that plant species had a pronounced effect on endophytic bacterial association and maximum endophytes (56.5%) were associated with T. domingensis. These endophytic bacteria mainly belonged to different species of the genera Bacillus (39%), Microbacterium (12%) and Halomonas (12%). Eight of the 41 strains showing maximum efficiency of textile effluent degradation also exhibited plant growth-promoting activities such as production of indole-3-acetic acid and siderophore, presence of 1-amino-cyclopropane-1-carboxylic acid deaminase, and solubilization of inorganic phosphorous. This is the first study describing the diversity and plant-beneficial characteristics of the textile effluent-degrading endophytic bacteria associated with wetland plants. T. domingensis showed better growth in textile effluent and also hosted maximum number of endophytic bacteria in roots and shoots. The interactions between T. domingensis and its associated endophytic bacteria could be exploited to enhance the efficiency of constructed wetlands during the remediation of industrial effluent.     

海南和两广地区柱花草炭疽菌遗传多态性的RAPD分析

利用8个随机引物对来自海南、广东和广西的114个柱花草胶孢炭疽菌(Colletotrichum gloeosporioides)的菌株进行了随机扩增多态性DNA(RAPD)分析, 扩增谱带大小0.2-3.0 kb。8个随机引物中以CW38114扩增的谱带重现性和稳定性最好, 共扩增出786条谱带, 其中多态性谱带558条。供试菌株扩增图谱在260-650 bp处有3条明显的共同特征谱带, 在650-3 000 bp之间的谱带显示较大的多态性差异。聚类结果表明: 供试菌株主要分为四大遗传类型, 其中第I类和第III类有两个亚类; 三省区的菌株遗传多态性丰富程度依次为海南>广西>广东, 并表现出地区性分布差异。研究结果还表明柱花草炭疽菌株表现出一定程度的专化寄生性。     

Chromosome numbers and mode of reproduction in Picrishieracioides s.l. (Asteraceae), with notes on some other Picristaxa

Chromosome numbers for 88 plants of Picris hieracioides s.l. originating from 32 localities from Austria, Andorra, Croatia, France, Germany, Hungary, Italy, Slovakia and Spain are reported. All analysed plants were diploid, with 2n=2x=10. In addition to P. hieracioides s.l., we also found diploidy (2n=2x=10) in P. nuristanica from Kirgizia and P. japonica from Japan. Our data confirmed previously published results in the literature. We also studied the mode of reproduction in all three taxa. Picris nuristanica and P. japonica s.l. were able to produce some seeds by selfing, and this is the first record of autogamy in this genus. In P. hieracioides s.l. only strict allogamy was found, demonstrating the presence of a self-incompatibility system in this species. Because apomixis, namely apospory, has previously been reported in P. hieracioides , castration experiments were performed in order to confirm or reject this report. However, no evidence of apomictic seed formation was found within this species. In the light of our results, P. hieracioides should be considered a strictly sexual taxon.     

海南和两广地区柱花草炭疽菌遗传多态性的RAPD分析

利用8个随机引物对来自海南、广东和广西的114个柱花草胶孢炭疽菌（Colletotrichum gloeosporioides）的菌株进行了随机扩增多态性DNA（RAPD）分析,扩增谱带大小0．2-3．0kb。8个随机引物中以CW38114扩增的谱带重现性和稳定性最好,共扩增出786条谱带,其中多态性谱带558条。供试菌株扩增图谱在260-650bp处有3条明显的共同特征谱带,在650—3000bp之间的谱带显示较大的多态性差异。聚类结果表明：供试菌株主要分为四大遗传类型。其中第Ⅰ类和第Ⅲ类有两个亚类;三省区的菌株遗传多态性丰富程度依次为海南〉广西〉广东,并表现出地区性分布差异。研究结果还表明柱花草炭疽菌株表现出一定程度的专化寄生性。     

Bacterial endophytes from arid land plants regulate endogenous hormone content and promote growth in crop plants: an example of Sphingomonas sp. and Serratia marcescens

The objective of the present study was to determine the potential plant growth-promoting action of bacterial endophytes isolated from arid land-dwelling plants under normal conditions. Overall, five bacterial endophytes LK11 (Sphingomonas sp. LK11), TP5 (Bacillus subtilis), MPB5.3 (B. subtilis subsp. Subtilis), S9 (B. subtilis subsp. Subtilis), and TP1 (Serratia marcescens) were evaluated based on morphological characteristics after isolation and purification. Phytohormonal analysis of these endophytes predicted indole acetic acid (IAA) production 12.31?±?0.45?, 6.8?±?0.59, and 10.5?±?1.02?μM/mL in the culture broths of LK11, MPB5.3, and TP1, respectively. Under controlled greenhouse conditions, these endophytes were inoculated into soybean, and their growth-promoting characteristics were compared with those of non-phytohormone-producing endophytes. In terms of plant growth promotion, among IAA-producing endophytes, LK11 and TP1 greatly improved physiological characteristics such as shoot/root length, fresh/dry weight, and chlorophyll contents. However, the non-phytohormone-producing endophytes TP5 and S9 did not show a growth-promoting effect. Based on these results, plants inoculated with LK11 and TP1 along with a control were subjected to endogenous hormonal analysis and showed a significant increase in abscisic acid (457.30–398.55 vs. 205.93 ng/g D.W.) and a decrease in jasmonic acid content (50.07–85.07 vs. 93.90 ng/g D.W.), respectively. Total gibberellin content was found to significantly increase in endophyte-inoculated plants (155.43–146.94?ng/g D.W.) as compared to that in controls (113.76 ng/g D.W.). In summary, bacterial endophytes might be used to enhance crop plant physiological characteristics isolated from arid land-inhabiting plants under normal conditions.     

Serologic survey for selected arboviruses and other potential pathogens in wildlife from Mexico.

During 1988 and 1989, a serologic survey of wildlife was conducted in northeastern Mexico to determine the presence, prevalence, and distribution of arboviruses and other selected disease agents. Eighty mammal specimens were tested. Antibodies to vesicular stomatitis-Indiana, Venezuelan equine encephalitis-Mena II, Rio Grande virus, and vesicular stomatitis-New Jersey were detected predominantly in small mammals. Deer and mouflon (Ovis musimon) had antibodies to bluetongue and epizootic hemorrhagic disease. Two species had serologic evidence of recent exposure to Francisella tularensis. A white-tailed deer (Odocoileus virginianus) had antibodies to Anaplasma marginale. All specimens tested for antibodies against Yersinia pestis and Brucella abortus were negative. Sera from 315 birds were tested for antibody against five equine encephalitis viruses and six avian pathogens. During 1988, antibodies to Venezuelan equine encephalitis-Mena II, Venezuelan equine encephalitis-TC83, St. Louis encephalitis, eastern equine encephalitis, and western equine encephalitis were detected in birds of several species. Antibodies to Pasteurella multocida and Newcastle disease virus were also detected. Birds from five species presented antibodies to Mycoplasma meleagridis. Specimens tested for M. gallisepticum, M. synoviae, and Chlamydia psittaci were negative. To the best of our knowledge, this survey represents the first serologic evidence of bluetongue, Cache Valley virus, epizootic hemorrhagic disease, Jamestown Canyon virus, vesicular stomatitis-Indiana, vesicular stomatitis-New Jersey, Rio Grande virus, and tularemia reported among wildlife in Mexico.     

Studies of Fruit Development of Cacao (Theobroma cacao) in Relation to Cherelle Wilt: II. Auxins and Development of the Seeds

The auxins of cacao seeds were examined in relation to seeddevelopment by the wheat straight growth and the first internodeassays after separation by filter paper chromatography. Therewere four consistent growth-active zones on the chromatograms:two acid growth promoters, one acid inhibitor, and a neutralgrowth promoter. An acid promoter (cacao auxin I) was shownby two-way chromatography and its activity in the oat curvaturetest to be similar to indole-3-acetic acid. Cacao auxin 1 occurredin roughly equal amounts in seeds of different ages throughoutthe wilting phase. The other growth substances detected on thechromatograms were unlikely to be associated with wilting phenomena. Three stages can be recognized in the growth of the perispermand endosperm. The only measurable differences between wiltingand healthy seeds was in the perisperm, which was smaller inthe wilting seeds during the second stage, 50 to 80 days fromfertilization. No differences could be found in the dimensionsof the wilting and healthy seeds. The observations on seed developmentare discussed in relation to growth of the fruit and cacao auxin1. A summary of the major steps in the development of the healthyand wilting seeded fruit and the hypothetical pathway by whichseedless and partially seeded fruits arise is presented.     

Structure of Alnus fruticosa Rupr. s. l. and its relationships with other taxa of subgenus Alnobetula (Ehrhart) peterman

The reliability of morphological parameters of Alnus fruticosa s. l. on the territory of Asian Russia was studied. It has been revealed that the species has a complex internal structure stipulated by the climatic and forest-growing conditions in different parts of the range. The results of phenotypic variability and sequences of internal transcribed spacer ITS1 of nuclear ribosomal DNA of the taxa of the subgenus Alnobetula indicate the species rank of Alnus viridis and subspecies rank of A. fruticosa, A. sinuata, and A. crispa with the priority species name A. crispa. A. maximowiczii, A. glutipes, A. kamtschatica, and A. mandshurica are the result of intraspecific variability of the polymorphic species A. fruticosa.     

瓦形细胞在广义锦葵科中的分布及其系统学意义

基于文献和作者的研究,该文讨论了瓦形细胞的识别、类型、变异及在广义锦葵科中的分布及其系统学意义。瓦形细胞是一种特殊射线细胞,呈不定的横列,常分散在横卧细胞之间,常无细胞内含物,其宽度远比横卧细胞为小,根据瓦形细胞与横卧细胞高度、宽度的相对大小,分为榴莲型、中间型和翅子树型3种类型,目前为止仅在广义锦葵科9个亚科中的7亚科39个现存属和5个化石属中发现;榴莲型、翅子树型和中间型瓦形细胞分别在22、20和5个属中发现,具有中间型的属同时具有榴莲型或/和翅子树型。仅翅子树型和中间型瓦形细胞在化石中发现,榴莲型可能比翅子树型古老。瓦形细胞在广义锦葵科中的分布,对研究一些类群的系统发育有意义。