Genetic Basis of Carnivorous Leaf Development

Plant carnivory is often manifested as dramatic changes in the structure and morphology of the leaf. These changes appear to begin early in leaf development. For example, the development of the Sarracenia purpurea leaf primordium is associated with the formation of an adaxial ridge, whose growth along with that of the leaf margin resulted in a hollow structure that later developed into a pitcher. In Nepenthes khasiana, pitcher formation occurs during the initial stages of leaf development, although this has not been shown at the primordial stage. The formation of the Utricularia gibba trap resulted from the growth of the dome-shaped primordium in both the longitudinal and transverse directions. Recent research has begun to unfold the genetic basis of the development of the carnivorous leaf. We review these findings and discuss them in relation to the flat-shaped leaves of the model plant Arabidopsis.

First Report of Coffee Canker Disease Caused by Ceratocystis fimbriata in China

Coffee (Coffea arabica L.) is one of the most important agricultural commodities in the world market. As an important cash crop in China, coffee is cultivated mainly in Yunnan and Hainan provinces. During October 2013 and September 2020, coffee trees showing typical dieback and wilt symptoms were found in Nanping town (N 22° 38', E 101° 0'), Pu’er, and Puwen town (N 22° 32', E 101° 4'), Xishuangbanna in Yunnan province, China. Symptomatic trees initially exhibited yellowing of foliage, expanding in size along the leaf margin, then became blighted and dry, and the internal xylem in main stem discolored brown to black. Infected trees eventually developed dieback and wilt. Disease incidence ranged from 10% to 22% and 25% to 40% of crown symptoms in the affected coffee trees. In extreme cases, 50% out of 380 trees were affected. The stems of coffee trees with typical symptoms were collected, and then the diseased tissues were surface disinfected with 75% ethanol for 30 s and 0.1% mercuric chloride (HgCl2) solution for 2 min, rinsed three times with sterile distilled water, plated onto potato dextrose agar (PDA) medium, and incubated at 25°C. After 6 days, fungal mycelium was observed growing from the tissue. Three isolates (C3-1, C3-2, and C3-2-1) were obtained by picking spore masses from the apices of perithecia and transferring them to malt extract agar (MEA) medium and incubated at 25°C for 10 days to observe the cultural features. In culture, colonies reaching 65 mm within 10 days, mycelium initially white, then becoming light blue-green. After 6 days of formation, perithecia were black, globose (123.8 - 173.4 μm × 138.2 - 180.6 μm), and showed a long black neck (414.2 - 650.0 μm). Ascospores with outer cell wall forming a brim, hat-shaped, accumulating in a mucilaginous mass at the tips of ostiolar hyphae (4.3 μm × 6.0 μm). Cylindrical endoconidia (14.1 - 45.2 μm × 3.5 - 5.7 μm) were hyaline. Chain of barrel-shaped conidia (6.6 - 10.2 μm × 6.8 - 8.8 μm) were found. Aleuroconidia (10.8 - 16.9 μm × 9.1 - 13.0 μm) were olive-brown, ovoid or obpyriform, and smooth. Morphological characteristics of the fungus were consistent with the description of Ceratocystis fimbriata Ellis & Halst. (Engelbrecht and Harrington 2005). The three isolates were used for molecular identification, and their genomic DNA was extracted using the chelex-100 method (Xu et al. 2020). The internal transcribed spacer (ITS) region of rDNA was sequenced using the procedures of Thorpe et al. (2005). Analysis of the ITS sequence data (GenBank accessions KY580836, KJ511480, and KJ511479) showed that the isolates were 100% homologous to isolates of C. fimbriata from Punica granatum, Camellia sinensis, and Cucumis sativus in China (GenBank accessions KY580891, KY580870, and MH535909, respectively) by BLAST analysis. Neighbor-joining (NJ) phylogenetic analysis was performed using MEGA 6.06 based on the ITS sequences. The three isolates were clustered on the same clade with other C. fimbriata isolates with a high bootstrap value (90%). Therefore, the fungus was identified as C. fimbriata based on both morphological and molecular characteristics. Pathogenicity of the three isolates was tested by inoculating one-year-old pot grown coffee seedlings (C. arabica) through drenching the loams with 30 ml spore suspension (1 × 106 spores/ml). Control plants were inoculated with 30 ml of sterile distilled water. The trees were kept in a controlled greenhouse at 25°C and watered weekly. One month after inoculation, all inoculated plants produced typical dieback and wilt symptoms, whereas the control trees showed no symptoms. The same fungus was isolated from the inoculated trees on PDA and identified as C. fimbriata according to the methods described above, and no fungal growth was observed in the controls, thus fulfilling the Koch's postulates. Coffee canker disease caused by C. fimbriata has been reported in Indonesia and Colombia (Marin et al. 2003). To our knowledge, this is the first report of C. fimbriata causing canker disease of coffee trees in China.

Myrcia longipetiolata (Myrtaceae), a New Species from the Atlantic Forest of Bahia, Brazil

Abstract— Myrcia longipetiolata, a new species of Myrcia, is here described and illustrated. With a phylogenetic hypothesis, we show that this species belongs to Myrcia clade 10, a morphologically recognized and narrowly distributed group of species, which was recently discovered. Myrcia longipetiolata resembles M. unana due to its long leaves, acute or acuminate leaf apex, cuneate leaf base, long petioles, and they share the same area of occurrence. Myrcia longipetiolata differs from M. unana by its larger leaves, larger number of leaf secondary veins, marginal vein more distant from the leaf margin, lanceolate bracteoles, smaller and truncate, rounded, or apiculate calyx lobes with whitish to yellowish trichomes, and smaller floral disc. Besides the phylogenetic placement of M. longipetiolata within the genus, morphological comparisons with related species and comments on its distribution, habitat, and conservation status are also provided.

NUMERICAL TAXONOMY OF GENUS FICUS L. 1753 (MORACEAE), WITH ADDITION NEW RECORD SPECIES TO EGYPT

The taxonomy of Ficus L., 1753 species is confusing because of the intense morphological variability and the ambiguity of the taxa. This study handled 36 macro-morphological characteristics to clarify the taxonomic identity of the taxa. The study revealed that Ficus is represented in the Egyptian gardens with forty-one taxa; 33 species, 4 subspecies and 4 varieties, and classified into five subgenera: Ficus Corner, 1960; Terega Raf., 1838; Sycomorus Raf., 1838; Synoecia (Miq.) Miq., 1867, and Spherosuke Raf.,1838; out of them seven were misidentified. Amongst, four new Ficus taxa were recently introduced to Egypt namely: F. lingua subsp. lingua Warb. ex De Wild. & T. Durand, 1901; F. pumila L., 1753; F. rumphii Blume, 1825, and F. sur Forssk., 1775. The application of the multivariate analyses in plant systematics namely the two-way clustering analysis and the principal component analysis revealed that the qualitative characters as the presence or absence of lateral peduncular or ostiolar bracts and the leaf margin delimit the differentiation of subgenera within genus Ficus. Whereas the qualitative characters of the leaf as leaf arrangement, lamina shape, length, ratio of length to width, base, apex, number of lateral veins, stipules and figs either pedunculate or sessile, shape, and width are significantly separating the species within the different sections. Seven different identification keys of the studied taxa based on the examined characters are provided. In addition, a diagrammatic key for all the studied taxa is given.

Auxin-driven ecophysiological diversification of leaves in domesticated tomato

The study of crop diversification has focussed mainly on the genetic changes underlying traits favoured by humans. However, the passage from natural habitats to agronomic settings probably operated changes beyond those comprising the classical domestication syndrome. A deeper understanding of these traits and their genetic signature would be valuable to inform conventional crop breeding and de novo domestication of crop wild relatives. Heterobaric leaves have bundle sheath extensions (BSEs) that compartmentalise the sub-stomatal cavity whereas homobaric leaves do not; BSE development is known to be controlled by the OBSCURAVENOSA (OBV) locus and the obv mutant lacks BSEs whereas leaves carrying the wild type allele have BSEs. Here we identify a non-synonymous amino acid change in the OBV gene that exists as a rare balanced polymorphism in the natural range of wild tomatoes, but has increased in frequency in domesticated tomatoes, suggesting that the latter diversified into heterobaric and homobaric leaf types. The mutation disrupts a C2H2 zinc finger motif in the OBV protein, resulting in the absence of BSEs in leaves and here we show that this and other pleiotropic effects, including changes in leaf insertion angle, leaf margin serration, minor vein density and fruit shape, are controlled by OBV via changes in auxin signalling. Loss of function of the transcriptional regulator AUXIN RESPONSE FACTOR (ARF4) also results in defective BSE development, revealing an additional component of a novel genetic module controlling aspects of leaf development important for ecological adaptation and subject to breeding selections.

First report of banana (Musa acuminate L. AAA Cavendish, cv. Formosana) leaf spot disease caused by Curvularia geniculata in China

Banana (Musa acuminate L.) is an important tropical fruit in China. During 2019-2020, a new leaf spot disease was observed on banana (M. acuminate L. AAA Cavendish, cv. Formosana) at two orchards of Chengmai county (19°48ʹ41.79″ N, 109°58ʹ44.95″ E), Hainan province, China. In total, the disease incidence was about 5% of banana trees (6 000 trees). The leaf spots occurred sporadically and were mostly confined to the leaf margin, and the percentage of the leaf area covered by lesions was less than 1%. Symptoms on the leaves were initially reddish brown spots that gradually expanded to ovoid-shaped lesions and eventually become necrotic, dry, and gray with a yellow halo. The conidia obtained from leaf lesions were brown, erect or curved, fusiform or elliptical, 3 to 4 septa with dimensions of 13.75 to 31.39 µm × 5.91 to 13.35 µm (avg. 22.39 × 8.83 µm). The cells of both ends were small and hyaline while the middle cells were larger and darker (Zhang et al. 2010). Morphological characteristics of the conidia matched the description of Curvularia geniculata (Tracy & Earle) Boedijn. To acquire the pathogen, tissue pieces (15 mm2) of symptomatic leaves were surface disinfected in 70% ethanol (10 s) and 0.8% NaClO (2 min), rinsed in sterile water three times, and transferred to potato dextrose agar (PDA) for three days at 28°C. Grayish green fungal colonies appeared, and then turned fluffy with grey and white aerial mycelium with age. Two representative isolates (CATAS-CG01 and CATAS-CG92) of single-spore cultures were selected for molecular identification. Genomic DNA was extracted from the two isolates, the internal transcribed spacer (ITS), large subunit ribosomal DNA (LSU rDNA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-alpha (TEF1-α) and RNA polymerase II second largest subunit (RPB2) were amplified and sequenced with universal primers ITS1/ITS4, LROR/LR5, GPD1/GPD2, EF1-983F/EF1-2218R and 5F2/7cR, respectively (Huang et al. 2017; Raza et al. 2019). The sequences were deposited in GenBank (MW186196, MW186197, OK091651, OK721009 and OK491081 for CATAS-CG01; MZ734453, MZ734465, OK091652, OK721100 and OK642748 for CATAS-CG92, respectively). For phylogenetic analysis, MEGA7.0 (Kumar et al. 2016) was used to construct a Maximum Likelihood (ML) tree with 1 000 bootstrap replicates, based on a concatenation alignment of five gene sequences of the two isolates in this study as well as sequences of other Curvularia species obtained from GenBank. The cluster analysis revealed that isolates CATAS-CG01 and CATAS-CG92 were C. geniculata. Pathogenicity assays were conducted on 7-leaf-old banana seedlings. Two leaves from potted plants were stab inoculated by puncturing into 1-mm using a sterilized needle and placing 10 μl conidial suspension (2×106 conidia/ml) on the surface of wounded leaves and equal number of leaves were inoculated with sterile distilled water serving as control (three replicates). Inoculated plants were grown in the greenhouse (12 h/12 h light/dark, 28°C, 90% relative humidity). Necrotic lesions on inoculated leaves appeared seven days after inoculation, whereas control leaves remained healthy. The fungus was recovered from inoculated leaves, and its taxonomy was confirmed morphologically and molecularly, fulfilling Koch’s postulates. C. geniculata has been reported to cause leaf spot on banana in Jamaica (Meredith, 1963). To our knowledge, this is the first report of C. geniculata on banana in China.

A revised infrageneric classification of Bellevalia Lapeyr. (Asparagaceae: Scilloideae) based on molecular analysis

Within the genus Bellevalia (Asparagaceae: Scilloideae: Hyacintheae), flower shape and colour, the ratio of leaf to scape length, and the orientation of the pedicels were traditionally used to delimit sections, subsections and species. We conducted a phylogenetic analysis of representative species from sections Bellevalia, Conicae, Nutantes and Oxydontae, representing four out of the six sections that are currently recognised, using the four chloroplast regions rbcL, matK, trnL intron, and trnL-F spacer. Our results indicate that the sections are not monophyletic as currently circumscribed. Our analyses retrieve two major, well-supported clades. The first clade (sect. Conicae) includes only species with ciliate leaves and green-veined perianth lobes, and the second clade (sect. Bellevalia) includes the species lacking these characteristics. Within the second clade, the species with yellow anthers are separated from those with violet anthers. Our molecular analysis does not support the traditional subdivisions of the genus but establishes the value of cilia on the leaf margin, green veins in the perianths lobes, and the colour of the anthers as indicators of relationships among the species. We recommend that the current four sections and six subsections of Bellevalia be reduced to two sections without subsections and we provide an updated and corrected nomenclature for these sections and subsections, designating lectotypes where necessary.

Leaf blight on Photinia × fraseri caused by Pestalotiopsis trachicarpicola in China

Photinia × fraseri is a well-known green plant mainly distributed in the Yangtze River and Yellow River Basin, east and southwest of China (Guan et al. 2013). In October 2020, typical leaf blight symptoms on roughly 10% leaves in a Photinia × fraseri shrub were observed in the campus of Sichuan Agricultural University (30°42′19″ N, 103°51′29″ E). Initially, chlorotic lesions with brown margins occurred on the leaf margin, then the large patches formed to cause leaves necrotic, finally lesions to dry and acervulus bred in 2–4 months later. Five single conidium isolates were carried out (Chomnunti et al. 2014) cultured on potato dextrose agar (PDA) at 25 ℃. All isolates shared similarly morphological characteristics, which was white and thin, and the reverse were yellowish. Mycelium was hyaline, sparsely septate, measuring 1–4 μm in diam. Conidiogenesis formed after 7 days. Conidiogenous cells were discrete, lageniform, smooth, thin-walled, colorless. Conidia were fusiform, straight to slightly curved, 4-septate, 21–30 × 5–7 μm (x ̅= 27 × 6.0 μm, n=30); basal cells were obconic with truncate base, hyaline, thin- and smooth-walled, 4–7 μm long (x ̅= 5.5 μm, n=30); three median cells were doliiform with thick walls, concolorous, olivaceous, constricted at the septa, and septa and periclinal walls were darker than the rest of the cell, 14–20 μm long (x ̅= 17 μm, n=30); apical cells were hyaline, conic to cylindrical, 3.0–6.5 μm long (x ̅= 4.5 μm, n=30), with 2–4 (mostly 3) tubular apical appendages arising from the upper portion, rarely branched, 7.5–18 μm long (x ̅= 12 μm, n=50); basal appendage was single, unbranched, 3–10 μm long (x ̅= 6.5 μm, n=30). DNA was extracted from the representative strain (SICAUCC 21-0012), and the internal transcribed spacer (ITS) region, the large subunit of the nrDNA (LSU), translation elongation factor 1-alpha (tef1-α), and partial sequences of β-tubulin (tub2) were amplified by polymerase chain reaction and sequenced with primers ITS5/ITS4, LR0R/LR5, 728F/1567R, and Bt2a/Bt2b, respectively (Zhang et al. 2012, Ariyawansa & Hyde 2018). The sequences were deposited in GenBank, viz. MZ453106, MZ453108, MZ467300, MZ467301, respectively. The nucleotide blast showed 99% (ITS, 0 gaps), 100% (tub2, 0 gaps), 100% (tef1-α, 0 gaps) identities with the ex-type Pestalotiopsis trachicarpicola Yan M. Zhang & K. D. Hyde (IFRDCC 2440). The fungus was identified as P. trachicarpicola combined with phylogeny and morphology (Maharachchikumbura et al. 2012, Zhang et al. 2012). To conduct Koch’s postulates, five healthy 6-year-old P. × fraseri were inoculated with 10 µl spore suspension (106 conidia/ml) onto the wounded sites (five leaves per plant, ~1 to 2 years old) via sterile pin, and five healthy plants treated with sterile dH2O as controls (Yang et al. 2021). The plants were placed in a greenhouse at 25°C with relative humidity >80%. After 2 months, leaf blight symptoms gradually emerged on inoculated leaves, and the controls were symptomless. Fungal isolates from symptomatic plants showed similar morphological characteristics as SICAUCC 21-0012, and the pathogen was not isolated from asymptomatic plants. To our knowledge, this is the first report of leaf blight caused by P. trachicarpicola on Photinia × fraseri in China. Disease management should be adopted properly to restore and improve its ornamental value.

Leaf geometric morphometric analyses of Callicarpa and Geunsia (Lamiaceae) in the Malesian region

Danila JS, Alejandro GJD. 2021. Leaf geometric morphometric analyses of Callicarpa and Geunsia (Lamiaceae) in the Malesian region. Biodiversitas 22: 4379-4390. Leaves are one of the most substantial organs of plants for it serves as a basis of species identification. Leaf morphology provides distinguishing features that help in the discrimination of plant species as well as investigation of leaf features among populations. This study aimed to investigate leaf shape variations between the two genera Geunsia Blume group and its closely related taxon, Callicarpa L. (Lamiaceae) using a landmark-based geometric morphometric method. The differences in the leaf shape among former members of Geunsia, namely C. apoensis, C. basilanensis, C. flavida, C. paloensis, C. pentandra, C. ramiflora, and C. surigaensis are also evaluated. Two primary landmarks and 14 semilandmarks were assigned in all samples to represent changes around the leaf margin. The Procrustes fit was generated using MorphoJ software which displays the mean and landmark position for individual configurations. Canonical Variate Analysis (CVA) and Mahalanobis Distance (MD) were able to discriminate all samples of Geunsia species using a scatter plot. Furthermore, Procrustes ANOVA showed a significant difference (P = 0.0082) among the seven species of the Geunsia group. Based on the results obtained, geometric morphometrics of leaf shape is effective in interspecific discrimination within members of Geunsia. However, the result of Discriminant Analysis (DA) showed that Geunsia and Callicarpa groups made leaf shape differences inefficient in discriminating the two genera. Therefore, further morphological studies on landmark-based geometric morphometrics of leaf shape involving a larger number of samples especially in the study of intergeneric classification are suggested.

First Report of Alternaria alternata Causing Leaf Spot on Menisper-mum dauricum DC. in China

Menispermum dauricum DC. is an ornamental plant used in traditional Chinese medicine. (Tang et al. 1992). In September 2019, a leaf spot on M. dauricum DC. was first found in a medicinal plant plantation in Harbin city (45.80°N, 126.53°E), Heilongjiang Province, China. The incidence was 76-90% on the 0.02 ha plantation. The initial symptoms were irregular black and brown spots on the leaves. The lesions expanded and coalesced, eventually leading to blight. Fresh leaf samples from ten M. dauricum plants with typical symptoms were collected. The areas of leaf between symptomatic and healthy tissue (5㎜×5㎜) were cut and surface disinfeated in 75% ethanol for 2 min, and with 1% HgCl2 for 1 min, and then rinsed three times with sterile water. Small lesion pieces were incubated on potato dextrose agar (PDA) for 7 days at 25℃, in the dark. Ten fungal isolates were obtained and transferred onto new PDA and potato carrot agar (PCA) plates to establish pure cultures. After 8 days, the colonies on PDA were 75-86㎜ in diameter, circular, with distinct concentric rings and a whitish aerial-mycelium margin, cottony, light gray to dark bluish brown. The colonies on the PCA were olive-green and bordered by white aerial hyphae. A total of 150 conidia were single or in short chains, obclavate, oval or inverted pear, light brown to brown, smooth or slightly spiny, with 1 to 6 transverse septa, 0 to 4 longitudinal or oblique septa, not narrow or slightly narrowed at the separation, 22.5-42.5×7.5-15.5㎛, and rostrate. Conidiophores were simple, erect, or ascending, dark brown, geniculate, septate, and with one or several conidial scars, 32.5-77.5×3.0-5.0㎛. Beaks were columnar or conical, 7.5-22.5×2.5-3.5㎛. Morphologically, all ten isolates were most similar to Alternaria alternata (Simmons 2007). For further identification of the fungus at the molecular level, internal transcribed spacer rDNA regions (ITS), RNA polymerase second largest subunit gene (RPB2) and Alternaria major allergen (Alt a 1) were amplified and sequenced using the primers ITS1 and ITS4, RPB2-5F2 and RPB2-7CR, Alt-for and Alt-rev (Woudenberg et al. 2015). The resulting sequences were deposited in GenBank (ITS: MT995193, MZ150794, RPB2: MT999483, MZ170963, Alt a 1: MT802122, MZ170962). BLAST search of these sequences showed 99%-100% homology with the ITS (FJ196306), RPB2 (KC584375) and Alt a 1 (KT315515) of the type strain CBS 916.96 of A. alternata, respectively. Thus, the fungus was identified as A. alternata based on the morphology and molecular analysis. For the pathogenicity test, spore suspensions (1×106 spores/mL) of the representative isolates BFG001 and BFG002 were sprayed onto the leaves of six healthy plants, separately. As a control, six plants were treated with sterile distilled water. The plants used in the experiment were covered with plastic bags and incubated at 25℃ for 10 days. Eight days after inoculation, irregular, slightly sunken black leaf spots appeared at the leaf margin. The experiment was repeated three times with the same method. The same fungus was successfully re-isolated from the leaves of the inoculated plants, fulfilling Koch’s postulates. No symptoms were observed on control plants. To our knowledge, this is the first report of leaf spot disease on M. dauricum DC. caused by A. alternata in the world. The appearance of leaf spot disease reduces the yield and quality of Chinese medicinal materials. This report has laid the foundation for the further research and control of leaf spot disease.