<Emphasis Type="Italic">Diaporthe</Emphasis> from walnut tree (<Emphasis Type="Italic">Juglans regia</Emphasis>) in China,with insight of the <Emphasis Type="Italic">Diaporthe eres</Emphasis> complex

Species of Diaporthe are important plant pathogenic fungi that commonly occur on a wide range of hosts. They are relatively difficult to identify due to their extreme similarity in morphology and confusing multigene phylogeny, especially in the Diaporthe eres complex. In the present study, isolates were collected from diseased branches of Juglans regia in China. Most strains were clustered into the D. eres species complex based on the combined internal transcribed spacer (ITS) region, partial calmodulin (CAL), histone H3 (HIS), translation elongation factor 1-alpha (TEF1-α) and beta-tubulin (TUB) genes. To focus on this complex, CAL, TEF1-α and TUB were selected in further phylogenetic analyses that showed a better topology compared with combined five-gene phylogeny. Results revealed that all strains which clustered in the Diaporthe eres complex from Juglans regia in China were Diaporthe eres. Results suggested a revised species criterion in the Diaporthe eres complex. The current study uncovered a new species here described as Diaporthe. tibetensis.     

Two novel Talaromyces species isolated from medicinal crops in Korea

Two novel biverticillate Talaromyces species, T. angelicus and T. cnidii, were collected from the medicinal crops Angelica gigas and Cnidium officinale, respectively, in Korea. Phylogenetic analyses with the nuclear ribosomal internal transcribed spacer (ITS) region and the β-tubulin gene as well as morphological analyses revealed that the two species differ from any known Talaromyces species. Talaromyces angelicus is related to T. flavovirens in the phylogeny of the ITS region, but the new species is grouped together with Penicillium liani and T. pinophilus in terms of its β-tubulin phylogeny, and its growth rate on Czapek yeast autolysate differs from that of T. flavovirens. Talaromyces cnidii is phylogenetically similar to T. siamensis, but exhibits differences in the morphologies of the colony margin, metulae, and conidia.     

In Vitro Photodynamic Inactivation of Plant-Pathogenic Fungi Colletotrichum acutatum and Colletotrichum gloeosporioides with Novel Phenothiazinium Photosensitizers

The increasing tolerance to currently used fungicides in both clinical and agricultural areas is of great concern. The nonconventional light-based approach of antimicrobial photodynamic treatment (APDT) is a promising alternative to conventional fungicides. We evaluated the effects of APDT with four phenothiazinium derivatives (methylene blue [MB], new methylene blue N [NMBN], toluidine blue O [TBO], and the novel pentacyclic phenothiazinium photosensitizer [PS] S137) on conidia of three fungal species (Colletotrichum acutatum, Colletotrichum gloeosporioides, and Aspergillus nidulans). The efficacy of APDT with each PS was determined, initially, based on photosensitizer MICs. Additionally, the effects of APDT with two selected PSs (NMBN and S137) on survival of conidia were evaluated. The subcellular localization of the PS in C. acutatum conidia was determined. The effects of photodynamic treatments on leaves of the plant host Citrus sinensis were also investigated. APDT with S137 showed the lowest MIC. MICs for S137 were 5 μM for the three fungal species when a fluence of 25 J cm⁻² was used. APDT with NMBN (50 μM) and S137 (10 μM) resulted in a reduction in the survival of the conidia of all species of approximately 5 logs with fluences of ≥15 J cm⁻². Washing of the conidia before light exposure did not prevent photodynamic inactivation. Both NMBN and S137 accumulated in cytoplasmic structures, such as lipid bodies, of C. acutatum conidia. No damage to orange tree leaves was observed after APDT.     

Population structure and genetic variation in the genus Dipteronia Oliv. (Aceraceae) endemic to China as revealed by cpSSR analysis

The genus Dipteronia Oliv. endemic to central and southern China consists of two species, D. sinensis Oliv. and D. dyerana Henry, both of them are rare and endangered. In the present study, genetic variation in 17 populations of D. sinensis and four populations of D. dyerana was estimated using ten polymorphic chloroplast microsatellite loci (cpSSR). Forty-nine chloroplast haplotypes were identified from 204 individuals analyzed. Thirty-nine haplotypes were found in D. sinensis, while ten in D. dyerana. No haplotype was shared between the species. AMOVA analysis revealed that the majority of the genetic variation was partitioned among populations within D. sinensis (F _ST = 0.7980) and D. dyerana (F _ST = 0.654). Cluster analysis grouped the 21 populations into two groups according to their species delimitation. The populations of D. sinensis were further divided into two subgroups corresponding to their geographical distributions. Correlation analysis revealed significant correlation between geographical distance and genetic distance of these populations (r = 0.326, p < 0.01 for D. sinensis vs. r = 0.777, p < 0.05 for D. dyerana). Genetic structure of these populations and a calculated pollen-to-seed flow ratio of (3.2:1 vs. 0.6:1) within the species suggested that little gene flow has occurred among the populations over an extended period. Thus, it implies that the genus Dipteronia might have experienced a genetic bottleneck and limited expansion during its evolutionary history.     

Evaluation of Diachasmimorpha longicaudata (Hymenoptera: Braconidae) as a mortality factor of Ceratitis capitata (Diptera: Tephritidae) infesting Citrus species under laboratory and field-cage conditions

Ceratitis capitata (or medfly) is one of the major pests currently affecting fruit crops in northwestern Argentinian Citrus-producing areas. Medfly populations are sustained in large exotic fruits, such as Citrus paradisi, Citrus aurantium and Citrus sinensis, which are known to hinder the activity of almost all native parasitoid species. Therefore, a feasible approach to controlling medfly involves the use of exotic parasitoids such as Diachasmimorpha longicaudata. In this study, the prediction that parasitoid females would be proficient at finding medfly larvae infesting the Citrus species mentioned earlier was tested. Particularly, the variation in fruit species preference by parasitoid females, the efficacy of the parasitoid to kill medfly and the effect of host density on parasitoid performance were determined. Parasitoids were allowed to forage for 8 h on grapefruits and oranges artificially infested with medfly larvae under controlled (laboratory) and uncontrolled (field cage) environmental conditions. Fruit choice and no-choice tests were performed. Results were assessed by comparing the number of female visits to and ovipositor insertions into the fruit, and parasitoid emergence, parasitism and additional host mortality percentages. Parasitoid preference for visiting larger fruits (sour orange and grapefruit) may be related to the increased fruit surface area. Ovipositional activity on fruit was influenced by the variation of the larval host density per unit of fruit surface. The higher parasitism rates recorded from sweet orange would be mainly a result of both increased host density and fruit physical features. Nevertheless, D. longicaudata showed the capacity to parasitise hosts in all Citrus species tested.     

Proteomic changes of Citrus roots in response to long-term manganese toxicity

Key message

Fifty-three and thirty-nine differentially expressed protein spots were isolated from Mn-toxic Citrus sinensis and Citrus grandis roots, respectively. Mn-toxicity-induced changes in protein profiles greatly differed between the two species.

Abstract

Limited information is available on the manganese (Mn)-toxicity-responsive proteins in plant roots. ‘Sour pummelo’ (Citrus grandis) and ‘Xuegan’ (Citrus sinensis) seedlings were irrigated for 17 weeks with 2 (control) or 600 μM (Mn-toxic) MnSO₄. C. sinensis displayed more tolerance to Mn-toxicity than C. grandis, which may be related to more Mn accumulation in roots and less Mn distribution in shoots. Using two-dimensional electrophoresis (2-DE), we isolated 11 up-regulated and 42 down-regulated protein spots from Mn-toxic C. sinensis roots, and 25 up-regulated and 14 down-regulated protein spots from Mn-toxic C. grandis roots. This indicated more metabolic flexibility in C. sinensis roots, thus contributing to the Mn-tolerance of C. sinensis. According to the biological functional properties, these differentially expressed proteins in the two species were classified into the following categories: protein metabolism, nucleic acid metabolism, carbohydrate and energy metabolism, stress responses, cell wall and cytoskeleton, cell transport, signal transduction and fatty acid metabolism. Under Mn-toxicity, proteins involved in nucleic acid metabolism, glycolysis and cell transport were up-regulated in nontolerant C. grandis roots, and down-regulated in tolerant C. sinensis roots. The notable down-regulation of proteins in Mn-toxic C. sinensis roots with less accumulation of carbohydrates may provide an advantage to the net carbon balance by lowering related metabolic processes, and enhancing the Mn-tolerance of C. sinensis. To conclude, there are many important differences in Mn-toxicity-induced changes in protein profiles and metabolic responses between the two species.     

Two new species of nematode-trapping fungi: relationships inferred from morphology,rDNA and protein gene sequence analyses

Two new nematode-trapping fungi, Dactylellina sichuanensis and D. varietas from China, which capture nematodes by both adhesive knobs and non-constricting rings, are described and illustrated. D. sichuanensis is characterized by both adhesive knobs and non-constricting rings, solitary conidiophores and 3-(4)-6 septate conidia, as compared with species such as D. appendiculata, D. candida, D. leptospora and D. lysipaga. Although D. sichuanensis shares similar types of trapping devices, the presence of simple conidiophores and spindle-shaped conidia with these species, it can be distinguished by its larger conidia and presence of more than four septa. In D. sichuanensis, a single conidium is born at the tip of conidiophore, while in D. candida, 3–10 conidia are born near the apex of conidiophore in a capitate arrangement. It produces spindle-shaped conidia with 3–6 septa, whereas D. leptospora produces cylindrical-shaped conidia with 5–15 septa. D. appendiculata captures nematodes by adhesive knobs whereas D. sichuanensis captures nematodes by both adhesive knobs and non-constricting rings. They also differ in conidial size (35–82.5 μm in D. sichuanensis as compared with 57–108 μm in D. appendiculata). D. varietas is characterized by conidiophores that are branched at right-angles, and elongate to fusoid conidia, with 7–8 septa (more than 25% of which are curved). D. varietas resembles D. asthenopaga, Dactylella oxyspora and Monacrosporium multiseptatum, but has elongate-fusoid conidia, whereas D. asthenopaga possesses obconical or clavate conidia. D. varietas forms both adhesive knobs and non-constricting rings whereas Dactylella oxyspora does not produce any trapping device. M. multiseptatum differs from D. varietas in having larger conidia with an inflated middle cell. Phylogenetic analyses based on nuclear and protein coding DNA sequences (18 S, and a combined 28 S + 5.8 S + β-tubulin dataset) indicate that these two taxa should be assigned to the family Orbiliaceae.     

Microscopical observation of inhibition-behaviors against Diaporthe citri by pre-treated with Pseudomonas putida strain THJ609-3 on the leaves of citrus plants

Citrus melanose is one of the most important diseases in orchards cultivating citrus in the world. Although the disease does not cause yield loss, the profitability of the infected fruits is often reduced in the fresh-market, resulting in economic loss. In this study, disease reduction was proven by pre-treatment with Pseudomonas putida strain THJ609-3. In order to illustrate mechanism of the disease reduction by the bacterial strain, the infection behaviors of Diaporthe citri and necrosis deposit of plant tissue were observed using a fluorescence microscope. On the leaves pre-treated with the strain THJ609-3, germination rates of D. citri conidia were significantly decreased compared to those of the untreated control. Scanning electron microscopical observations showed that bacterial cells were attached to the surface of fungal hyphae. Furthermore, morphological change of germ tubes of the conidia was detected. These results suggest that the disease reduction may be caused by the direct antifungal activity of the bacterial strain on the leaf surfaces.     

Preference and performance of Diachasmimorpha kraussii (Fullaway) (Hymenoptera: Braconidae) on five commercial fruit species

Diachasmimorpha kraussii is a polyphagous endoparasitoid of dacine fruit flies. The fruit fly hosts of D. krausii, in turn, attack a wide range of fruits and vegetables. The role that fruits play in host selection behaviour of D. kraussii has not been previously investigated. This study examines fruit preference of D. kraussii through a laboratory choice‐test trial and field fruit sampling. In the laboratory trial, oviposition preference and offspring performance measures (sex ratio, developmental time, body length, hind tibial length) of D. kraussii were investigated with respect to five fruit species [Psidium guajava L. (guava), Prunis persica L. (peach), Malus domestica Borkh. (apple), Pyrus communis L. (pear) and Citrus sinensis L. (orange)], and two fruit fly species (Bactrocera jarvisi and B. tryoni). Diachasmimorpha kraussii responded to infested fruit of all fruit types in both choice and no‐choice tests, but showed stronger preference for guava and peach in the choice tests irrespective of the species of fly larvae within the fruit. The wasp did not respond to uninfested fruit. The offspring performance measures differed in a non‐consistent fashion between the fruit types, but generally wasp offspring performed better in guava, peach and orange. The offspring sex ratio, except for one fruit/fly combination (B. jarvisi in apple), was always female biased. The combined results suggest that of the five fruits tested, guava and peach are the best fruit substrates for D. krausii. Field sampling indicated a non‐random use of available, fruit fly infested fruit by D. kraussii. Fruit fly maggots within two fruit species, Plachonia careya and Terminalia cattappa, had disproportionately higher levels of D. krausii parasitism than would be expected based on the proportion of different infested fruit species sampled, or levels of fruit fly infestation within those fruit.     

Morphological and genomic evidence for a new species of Corallorhiza (Orchidaceae: Epidendroideae) from SW China

Corallorhiza sinensis, a new species of mycoheterotrophic orchid from western Sichuan, China, is described and illustrated based on molecular and morphological evidence. It is morphologically similar to Corallorhiza trifida, but can be distinguished by bigger flowers, both sepals and petals with 3 veins, and longer lateral lobes of lip. To distinguish the new Corallorhiza species and explore its phylogenetic position within subtribe Calypsoinae, this study employed sequences of the nuclear ribosomal DNA (nrDNA) and whole plastome assembled from the genome skimming approach. The plastome is 148,124 bp in length, including a pair of inverted repeats (IRs) of 26,165 bp, a large single-copy region (LSC) of 82,207 bp, and a small single-copy region (SSC) of 13,587 bp. Further, phylogenetic analyses were performed using nrDNA sequence and 79 coding sequences (CDSs) from 26 complete plastomes of subtribe Calypsoinae. The phylogenetic tree based on nrDNA sequence suggested that Corallorhiza is a monophyletic group, and strongly support C. sinensis as sister to the rest species of Corallorhiza. The plastid tree showed that 10 Corallorhiza species grouped into two clades and C. sinensis is most closely related to the North American C. striata and C. bentleyi instead of Oreorchis foliosa and O.angustata in the same clade. The plastid tree and nrDNA tree indicate Oreorchis is a paraphyletic. Although the topological conflicts are displayed between plastome and nrDNA phylogenies of C. sinensis, it is still the most closely related to Corallorhiza. Comparative analysis showed that C. sinensis populations are characteristic of the intermediate morphological traits between Corallorhiza and Oreorchis. The finding of this new species from China shed new light on the phylogeny of Oreorchis and Corallorhiza.     

Morpho-Molecular and Ultrastructural Characterization of Discocriconemella Parasinensis n. Sp. from Zhejiang Province,China

During a recent inventory survey of the nematofauna of tea plantation at Zhejiang Province, China, Discocriconemella parasinensis n. sp. was detected in the rhizosphere of Camellia sinensis. The new species can be characterized by having the uninterrupted rounded labial disc, en face view showing rectangular-rounded labial plate without submedian lobes, R = 82.6 (80–86), Rex = 22 (21–23), stylet length of 68.3 (59–76) μm, excretory pore located 1–2 annuli posterior to the esophageal bulb, vulva open, postvulval body elongated conoid, and tail conoid with bilobed terminus. Morphologically, the species shares the same lip-type with D. discolabia, D. mauritiensis, D. mineira, D. perseae, and D. sinensis. Phylogenetic relationships of the new species based on D2–D3 expansion segments of 28S, ITS, and 18S rRNA genes revealed that D. parasinensis n. sp. formed a separated clade from other criconematid species, thereby supporting its status as a new species of the genus. The new species showed close phylogenetic relationships with Criconemoides geraerti.     

Morphology,morphogenesis and molecular phylogeny of Lamtostyla granulifera sinensis subsp. nov. (Ciliophora,Hypotrichia) from a wetland in China

The morphology, morphogenesis and molecular phylogeny of a hypotrichous ciliate, Lamtostyla granulifera sinensis subsp. nov., isolated from northern China, were investigated. This population appeared highly similar in morphology to L. granulifera Foissner, 1997. However, on detailed investigation some non-overlapping features were identified, i.e., the body shape and the arrangement of the cortical granules. These differences suggested the separation at subspecies level. Furthermore, the morphogenesis of the new subspecies is described, which is characterized by: (1) the posterior part of the parental adoral zone of membranelles is renewed; (2) the amphisiellid median cirral row is formed from two anlagen; and (3) the frontoventral-transverse cirral anlagen II to VI generate one transverse cirrus each. Phylogenetic analyses based on SSU rDNA sequence data show that Lamtostyla species are scattered in different clades. The monophyly of the genus Lamtostyla is also rejected by the AU test in this study.     

Structural organization,classification and phylogenetic relationship of cytochrome P450 genes in Citrus clementina and Citrus sinensis

The genus Citrus is an important fruit crop and nutritional source for the good health of humans. Cytochrome P450s represent about 1 % of the proteome and mediate diverse biochemical reactions pertaining to both primary and secondary metabolism. Analysis of Citrus genomic resources identified 296 plant cytochrome P450s (CYP) coding genes in Citrus clementina, 272 in double haploid (dh) Citrus sinensis, and 202 in C. sinensis. In C. clementina and dh C. sinensis, CYP genes are distributed into nine clans. In the three genomes, single intron containing CYP genes are predominant in the A-type families. Among non-A-type CYP families, multiple intron containing genes are predominant. More number of genes in CYP A-type families over non-A-type families is attributed to rapid evolution of A-type genes facilitated by their gene organization. Further, complex gene organization of non-A-type genes with the presence of multiple introns might have contributed to the slower evolvement of paralogs. Majority of introns (1,660) from three genomes showed canonical GT-AG splice sites. However, 33 introns showed non-conventional GC… PyAG splice sites and functionality of these splice sites is confirmed by the ESTs lacking this intron. Across the families, gene organization is conserved between the three genomes. In dh C. sinensis, 22 genes were identified to have alternate splicing. Examination of scaffolds in C. clementina revealed that majority of the Citrus CYP genes are solitary and a few of them are in clusters of 3–8 genes. PCR amplification of C. sinensis genomic DNA with gene-specific primers failed to amplify out-grouped genes Ccl-CYP706A16 and Ccl-CYP706B1, confirming that they are specific to C. clementina. Differential number of CYP genes observed between C. clementina and C. sinensis is attributed to the extent of variability between their parents representing ancestral taxa.     

Trapping tephritid fruit flies (Diptera: Tephritidae) in citrus groves of Fujian Province of China

Certain tephritid fruit flies, such as the oriental fruit fly, Bactrocera dorsalis, the Chinese citrus fly, B. minax and the Japanese orange fly, B. tsuneonis (Diptera: Tephritidae: Dacinae), are destructive citrus pests in China. A two-year trapping study was conducted in pomelo, Citrus maxima, groves in Fujian Province of China. The objectives of this study were to investigate the species, the abundance of tephritid fruit flies in the orchards, as well as the efficacy of the selected lure traps to these flies. Four lure traps or devices, i.e. methyl eugenol + Steiner trap (ST), cuelure + ST, ammonium acetate + putrescine + ST, and sticky spheres, were deployed from June to November 2017 and April to October 2018. Six economically significant Dacini pests were trapped during the period. These flies are B. dorsalis, the melon fruit fly, Zeugodacus cucurbitae, the pumpkin fruit fly, Z. tau, the Malaysian fruit fly, B. latifrons, and other two species - B. rubigina and Z. scutellatus. B. dorsalis was the most abundant, accounting for more than 50% of the capture, followed by Z. cucurbitae. The remaining four species accounted for less than 2% of the total capture. B. minax and B. tsuneonis, two destructive citrus-damaging tephritid fruit flies in China, were not found during the trapping period. Methyl eugenol trapped the highest number of fruit flies, followed by cuelure.     

Kinetic and regulatory properties of phenylalanine ammonia-lyase from Citrus sinensis

• 1.1. The kinetic and regulatory properties of phenylalanine ammonia-lyase from Citrus sinensis fruit tissue were investigated. The substrate specificity of the enzyme was determined as well as the effects of pH and temperature on the catalytic activity.
• 2.2. The enzyme exhibits negative homotropic effects between the substrate binding centra.
• 3.3. Binding of l-phenylalanine to the enzyme is characterized by two K_m-values; K_m^L = 13 μM and K_m^H = 52 μM; with a Hill-interaction coefficient of 0.75.
• 4.4. The enzyme is subject to product inhibition by trans-cinnamate, but the effects of allosteric effectors and inhibitors seem to be of much greater importance in the short-term regulation of phenylpropanoid metabolism in Citrus sinensis.
• 5.5. The enzyme activity was found to be modulated by end-products of diverging metabolic pathways, viz. umbelliferone, scopoletin, naringenin, quercetin, kaempferol, benzoic acid and gallic acid.

     

Cellulase Activity, Endogenous Abscisic Acid, and Ethylene in Four Citrus Cultivars during Maturation

At maturity, the fruit of two early maturing orange cultivars, Hamlin and Pineapple (Citrus sinensis [L.] Osbeck), contained more ethylene and abscisic acid than the late maturing Valencia and Lamb Summer (C. sinensis [L.] Osbeck) cultivars. Ethylene (up to 95 nl/l in internal atmosphere) and abscisic (50 μg/kg dry weight flavedo) increased most rapidly in Pineapple, leading to increased cellulase activity and loosening of the fruit. Fruit of the two late maturing cultivars contained less than 25 nl/1 ethylene and 40 μg abscisic acid/kg dry weight of flavedo at peak maturity. Cellulase activity and loosening of the fruit of these late maturing cultivars was slight.