The role of pregrowth and preculture treatments in terms of both medium composition and exposure duration on survival of embryonic axes of Citrus madurensis after cryopreservation using the vitrification procedure was investigated. The optimal pregrowth treatment for excised embryonic axes was a 3-day treatment with 0.1M sucrose. Preculture was also essential in increasing survival after cryopreservation. Among the various media and treatment durations evaluated, a 24h-preculture of embryonic axes on medium with 0.3M sucrose and 0.5M glycerol was found to be optimal. Using these pregrowth and preculture conditions followed by treatment at 25 degrees C for 20 min each with a loading solution (0.4M sucrose + 2.0M glycerol) and then the PVS2 vitrification solution, direct immersion in liquid nitrogen, rapid rewarming, unloading in a 1.2M sucrose solution for 20 min and transfer of embryonic axes on recovery medium, 82.5% survival and regrowth without intermediary callus formation were obtained with C. madurensis embryonic axes.
In this paper, we demonstrate that C. madurensis embryonic axes can withstand cryopreservation using the encapsulation-dehydration technique. Up to 57.5 % survival was achieved using a standard encapsulation-dehydration protocol, which included pregrowth of encapsulated axes for 16 h in medium containing 0.8 M sucrose + 1 M glycerol, desiccation of beads to around 30 % moisture content (fresh weight basis) followed by rapid freezing. A slightly higher survival percentage (65 %) was obtained using a modified encapsulation-dehydration protocol, which included pretreatment of axes with 2 M glycerol + 0.6 M sucrose for 1 h, concomitantly with their encapsulation in 3 % calcium alginate beads, followed by desiccation of the beads to around 30 % moisture content.
This paper investigates the importance of loading and treatment with a vitrification solution on the survival of Citrus madurensis embryonic axes cryopreserved using a vitrification protocol. Among the seven different loading solutions tested, the solution containing 2 M glycerol + 0.4 M sucrose was the most efficient. Of the six vitrification solutions tested, the PVS2 vitrification solution, applied for 20 min at 25 degree C or for 60 min at 0 degree C, ensured the highest survival. A three-step vitrification protocol, involving the treatment of embryonic axes at 0 degree C with half strength PVS2 solution for 20 min then with full strength PVS2 for an additional 40 min was more efficient than a two-step protocol that involved treatment of axes directly with full strength PVS2 solution for 60 min. After rapid immersion in liquid nitrogen, rapid rewarming, unloading in a 1.2 M sucrose solution for 20 min, culture on solid medium with 0.3 M sucrose for 1 day and growth recovery for 4 weeks on standard medium, survival of C. madurensis embryonic axes reached 85 % following the three-step process, compared with 70 % for the two-step process.
This paper focuses on the evaluation of the k0 method of instrumental neutron activation analysis in biological materials. The method has been applied in multielement analysis of human hair standard reference materials from IAEA, No. 085, No. 086 and from NIES (National Institute for Environmental Sciences) No. 5. Hair samples from people resident in different parts of Malaysia, in addition to a sample from Japan, were analyzed. In addition, human kidney stones from members of the Malaysian population have been analyzed for minor and trace elements. More than 25 elements have been determined. The samples were irradiated in the rotary rack (Lazy Susan) at the TRIGA Mark II reactor of the Malaysian Institute for Nuclear Technology and Research (MINT). The accuracy of the method was ascertained by analysis of other reference materials, including 1573 tomato leaves and 1572 citrus leaves. In this method the deviation of the 1/E1+ alpha epithermal neutron flux distribution from the 1/E law (P/T ratio) for true coincidence effects of the gamma-ray cascade and the HPGe detector efficiency were determined and corrected for.
Rhipicephalus (Boophilus) microplus serves as an important ectoparasite of livestock and a vector of several pathogens resulting in diseases, subsequently affecting the agricultural field as well as the economy. The extensive use of synthetic acaricides is known to cause resistance over time and therefore a much safer, effective and environmentally friendly alternative to overcome tick infestation should be implemented. Larval immersion tests (LIT) were done to evaluate the effects of Citrus hystrix (Family: Rutaceae) and Cymbopogon citratus (Family: Poaceae) essential oils (EOs) for their individual and combined (1:1) acaricidal activity against the cattle tick. Results showed that LC50 and LC90 values in 24 and 48 hours for Cit. hystrix EO were 11.98% and 24.84%, and 10.95% and 21.71% respectively. LC50 and LC90 values for Cym. citratus EO were 1.21% and 6.28%, and 1.05% and 6.12% respectively. The mixture of EOs from two plants in 1:1 ratio (Cit. hystrix 50%: Cym. citratus 50%) was found to exhibit antagonistic effect (synergistic factor < 1). The 24 hours and 48 hours LC50 and LC90 values for combined EOs were 1.52% and 2.84%, and 1.50% and 2.76% respectively. Individual and combined essential oils were subjected to qualitative analysis using gas chromatography-mass spectrometry (GC-MS) to screen the chemical components present in EOs. Our results showed that the combination of Cit. hystrix and Cym. citratus at 1:1 ratio resulted in an antagonistic effect and the use of Cym. citratus alone is more toxic to R. (B.) microplus, making it a better alternative to chemical based acaricide.
Colorectal cancer is the second leading cause of cancer-related deaths in the United States. Recent studies prove that though chemotherapeutic agents are being used for the treatment of colon cancer, they become non-effective when the cancer progresses to an invasive stage. Since consumption of certain dietary agents has been linked with various cancers, fruit juices have been investigated for their consistently protective effect against colon cancer. The unique biochemical composition of fruit juices is responsible for their anticancer properties. In this review, the chemo-preventive effect of fruit juices such as pomegranate and citrus juices against colon cancer are discussed. For this purpose, the bioavailability, in vitro and in vivo effects of these fruit juices on colorectal cancer are highlighted. Moreover, there is a scarcity of studies involving human trials to estimate the preventive nature of these juices against colon cancer. This review will support the need for more preclinical tests with these crude juices and their constituents in different colorectal cancer cell lines and also some epidemiological studies in order to have a better understanding and promote pomegranate and citrus juices as crusaders against colon cancer.
Volatile compounds in the peel of calamansi (Citrus microcarpa) from Malaysia, the Philippines and Vietnam were extracted with dichloromethane and hexane, and then analysed by gas chromatography-mass spectroscopy/flame ionisation detector. Seventy-nine compounds representing >98% of the volatiles were identified. Across the three geographical sources, a relatively small proportion of potent oxygenated compounds was significantly different, exemplified by the highest amount of methyl N-methylanthranilate in Malaysian calamansi peel. Principal component analysis and canonical discriminant analysis were applied to interpret the complex volatile compounds in the calamansi peel extracts, and to verify the discrimination among the different origins. In addition, four common hydroxycinnamic acids (caffeic, p-coumaric, ferulic and sinapic acids) were determined in the methanolic extracts of calamansi peel using ultra-fast liquid chromatography coupled to photodiode array detector. The Philippines calamansi peel contained the highest amount of total phenolic acids. In addition, p-Coumaric acid was the dominant free phenolic acids, whereas ferulic acid was the main bound phenolic acid.
Solid-phase microextraction (SPME) coupled to gas chromatography has been applied for the headspace analysis (HS) of 12 target flavour compounds in a model orange beverage emulsion. The main volatile flavour compounds studied were: acetaldehyde, ethyl acetate, alpha-pinene, ethyl butyrate, beta-pinene, myrcene, limonene, gamma-terpinene, octanal, decanal, linalool and citral (neral plus geranial). After screening the fibre type, the effect of other HS-SPME variables such as adsorption temperature (25-55 degrees C), extraction time (10-40 min), sample concentration (1-100% w/w), sample amount (5-10 g) and salt amount (0-30% w/w) were determined using a two-level fractional factorial design (2(5-2)) that was expanded further to a central composite design. It was found that an extraction process using a carboxen-polydimethylsiloxane fibre coating at 15 masculineC for 50 min with 5 g of diluted emulsion 1% (w/w) and 30% (w/w) of sodium chloride under stirring mode resulted in the highest HS extraction efficiency. For all volatile flavour compounds, the linearity values were accurate in the concentration ranges studied (r(2) > 0.97). Average recoveries that ranged from 90.3 to 124.8% showed a good accuracy for the optimised method. The relative standard deviation for six replicates of all volatile flavour compounds was found to be less than 15%. For all volatile flavour compounds, the limit of detection ranged from 0.20 to 1.69 mg/L.
The constituents in a food emulsion interact with each other, either physically or chemically, determining the overall physico-chemical and organoleptic properties of the final product. Thus, the main objective of present study was to investigate the effect of emulsion components on beverage emulsion properties.
Au-Ag alloy nanoparticles are physically synthesized using rapid, simple and efficient Q-switched Nd:YAG pulsed laser ablation in liquid technique (PLAL). Au and Ag colloidal solutions are separately prepared by 1064 nm laser ablation of metallic target (gold and silver) which is immersed in deionized water. Au-Ag alloy nanoparticles are prepared by irradiating the mixture of Au and Ag colloidal solutions with 532 nm of second harmonic wavelength of Nd:YAG laser at three different ratio, 3:1, 1:1 and 1:3 within different exposure times. The three of plasmon absorption bands of Au-Ag nanoparticles are shifted linearly to the lower wavelength [499.67 nm (3:1), 481.25 nm (1:1), 467.91 nm (1:3)], as compared to plasmon absorption spectra of pure Au (520 nm) and Ag (400 nm). Moreover, the change in colors are also observed from red (Au) and yellow (Ag) to orange, brown and green color due to the Au-Ag alloy formations, respectively. Transmission electron microscopy shows the Ag shell around the inner core of Au spherical metal with broad size distribution due to the three different volume ratio, respectively (1.7 nm, 0.7 nm, 1.4 nm). Energy-dispersive X-ray spectroscopy analysis confirms the presence of Au and Ag elements in Au-Ag alloy nanoparticles without any contaminations. Attenuated total reflectance fourier transform infrared spectroscopy analysis also confirms the homogenous Au-Ag alloys chemical bonding.
A new genus of onchidiid slugs, Wallaconchis Goulding & Dayrat, gen. n., is described, including ten species. Five species were previously described but known only from the type material: Wallaconchis ater (Lesson, 1830), W. graniferum (Semper, 1880), W. nangkauriense (Plate, 1893), W. buetschlii (Stantschinsky, 1907), and W. gracile (Stantschinsky, 1907), all of which were originally classified in Onchidium Buchannan, 1800. Many new records are provided for these five species, which greatly expand their known geographic distributions. Five species are new: Wallaconchis achleitneri Goulding, sp. n., W. comendadori Goulding & Dayrat, sp. n., W. melanesiensis Goulding & Dayrat, sp. n., W. sinanui Goulding & Dayrat, sp. n., and W. uncinus Goulding & Dayrat, sp. n. Nine of the ten Wallaconchis species are found in the Coral Triangle (eastern Indonesia and the Philippines). Sympatry is high, with up to six species found on the island of Bohol (Philippines) and eight species overlapping in northern Sulawesi (Indonesia). Wallaconchis is distinguished from other onchidiids by its bright dorsal colors (red, yellow, orange) but those are extremely variable and not useful for specific identification. Internally, the reproductive system can be used to identify all Wallaconchis species. The copulatory organs of Wallaconchis species are especially diverse compared to other onchidiid genera, and the possible role of reproductive incompatibility in species diversification is discussed. All specimens examined were freshly collected for the purpose of a worldwide revision of the Onchidiidae Rafinesque, 1815. The species are well delineated using DNA sequences and comparative anatomy. Mitochondrial DNA analysis yields thirteen molecular units separated by a large barcode gap, while nuclear DNA yields nine units. By integrating nuclear DNA and mitochondrial DNA with morphology, ten species are recognized. The natural history of each species (e.g., the microhabitat where they are found) is also documented. Nomenclature is addressed thoroughly (the types of all onchidiid species were examined, lectotypes were designated when needed, nomina dubia are discussed). Morphological characters, transitions to new microhabitats, and diversification processes are discussed in the context of a robust molecular phylogeny.
Serving raw oysters with lemon juice is a delicacy in many restaurants in
Malaysia. Oysters (Crassostrea virginica) live in the seacoast and they share the same
environment as Vibrio parahaemolyticus. Consumption of raw oysters contaminated with V.
parahaemolyticus can lead to severe gastroenteritis. A study was performed to determine
whether lemon (Citrus limon) juice is able to inhibit the growth of V. parahaemolyticus after
being inoculated in raw oysters. Methods: Frozen oysters bought from a local supplier
weighing 6 g each were minced and placed in two bottles using sterile technique.
Approximately 1 ml of 107 CFU of V. parahaemolyticus (ATCC strain 17802) was added and
mixed in both bottles. The mixture was treated with 1 ml of lemon juice in only one of the
bottles and the other bottle served as a control. At every 30 s intervals for 2 min, 1 g of the
sample was taken for enumeration of viable cells onto thiosulphate citrate bile salt sucrose
(TCBS). Results: After 30 s of treatment with the lemon juice, it was observed that the
number of colonies in the treated samples reduced from 7 Log to 3 Log. Subsequently, no
viable V. parahaemolyticus was seen. It was also observed that there were 3 Log reductions
of V. parahaemolyticus after 30 s in untreated samples, however the number of colonies
remained stable until the end of the experiment. Conclusion: This study therefore shows
that lemon juice has some antimicrobial effect on V. parahaemolyticus in raw oysters.
With varied, brightly patterned wings, butterflies have been the focus of much work on the evolution and development of phenotypic novelty. However, the chemical structures of wing pigments from few butterfly species have been identified. We characterized the orange wing pigments of female Elymnias hypermnestra butterflies (Lepidoptera: Nymphalidae: Satyrinae) from two Southeast Asian populations. This species is a sexually dimorphic Batesian mimic of several model species. Females are polymorphic: in some populations, females are dark, resemble conspecific males, and mimic Euploea spp. In other populations, females differ from males and mimic orange Danaus spp. Using LC-MS/MS, we identified nine ommochrome pigments: six from a population in Chiang Mai, Thailand, and five compounds from a population in Bali, Indonesia. Two ommochromes were found in both populations, and only two of the nine compounds have been previously reported. The sexually dimorphic Thai and Balinese populations are separated spatially by monomorphic populations in peninsular Malaysia, Singapore, and Sumatra, suggesting independent evolution of mimetic female wing pigments in these disjunct populations. These results indicate that other butterfly wing pigments remain to be discovered.
Enhanced red and orange fluorescence emissions of Sm3+ Rare earth (RE) ions were observed in sodium‑zinc tellurite glasses embedded with silver and gold nanoparticles (NPs). The fine distribution of NPs in the glass matrix with an average diameter ~ 11.09 nm and ~3.86 nm for Ag and Au NPs respectively were confirmed by using transmission electron microscope (TEM). The embedding of Ag and Au NPs into the glass structure caused an increasing in the transition emission intensity of Sm3+ ions, which is ascribed to the progress of the presence of the localized surface Plasmon resonance (LSPR) indicating from the characteristic absorption peaks. The luminescence and absorption spectra have been discussed using a standard hypothesis Judd-Ofelt theory for a certain absorption transitions 6P3/2, 4I11/2, 6F11/2, 6F9/2, 6F7/2, 6F5/2, 6F3/2, 6H15/2, 6F1/2 and emission transitions 6H5/2, H7/2, 6H9/2 and H11/2 under 409 nm excitation of the Sm3+ ions. The decay life time curve exhibited a non-exponential behavior of the studied glass samples and the results were compared with the similar reported glasses. An efficient red and orange fluorescence emission illustrate that the Sm3+-doped sodium‑zinc tellurite embedded with Ag and Au NPs are potential materials for the laser illumination.
Symptoms of gray leaf spot were first observed in June 2011 on pepper (Capsicum annuum) plants cultivated in the Cameron Highlands and Johor State, the two main regions of pepper production in Malaysia (about 1,000 ha). Disease incidence exceeded 70% in severely infected fields and greenhouses. Symptoms initially appeared as tiny (average 1.3 mm in diameter), round, orange-brown spots on the leaves, with the center of each spot turning gray to white as the disease developed, and the margin of each spot remaining dark brown. A fungus was isolated consistently from the lesions using sections of symptomatic leaf tissue surface-sterilized in 1% NaOCl for 2 min, rinsed in sterile water, dried, and plated onto PDA and V8 agar media (3). After 7 days, the fungal colonies were gray, dematiaceous conidia had formed at the end of long conidiophores (19.2 to 33.6 × 12.0 to 21.6 μm), and the conidia typically had two to six transverse and one to four longitudinal septa. Fifteen isolates were identified as Stemphylium solani on the basis of morphological criteria described by Kim et al. (3). The universal primers ITS5 and ITS4 were used to amplify the internal transcribed spacer region (ITS1, 5.8, and ITS2) of ribosomal DNA (rDNA) of a representative isolate (2). A 570 bp fragment was amplified, purified, sequenced, and identified as S. solani using a BLAST search with 100% identity to the published ITS sequence of an S. solani isolate in GenBank (1). The sequence was deposited in GenBank (Accession No. JQ736024). Pathogenicity of the fungal isolate was tested by inoculating healthy pepper leaves of cv. 152177-A. A 20-μl drop of conidial suspension (105 spores/ml) was used to inoculate each of four detached, 45-day-old pepper leaves placed on moist filter papers in petri dishes (4). Four control leaves were inoculated similarly with sterilized, distilled water. The leaves were incubated at 25°C at 95% relative humidity for 7 days. Gray leaf spot symptoms similar to those observed on the original pepper plants began to develop on leaves inoculated with the fungus after 3 days, and S. solani was consistently reisolated from the leaves. Control leaves did not develop symptoms and the fungus was not reisolated from these leaves. Pathogenicity testing was repeated with the same results. To our knowledge, this is the first report of S. solani causing gray leaf spot on pepper in Malaysia. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) M. P. S. Camara et al. Mycologia 94:660, 2002. (3) B. S. Kim et al. Plant Pathol. J. 15:348, 1999. (4) B. M. Pryor and T. J. Michailides. Phytopathology 92:406, 2002.
This work studied the effectiveness of gaseous ozone disinfection on pummelo (Citrus Grandis L. Osbeck) fruit juice components. Unfiltered and filtered pummelo fruit juices were treated with gaseous ozone for up to 50 min with ozone concentration fixed at 600 mg/h. A microbiological and physicochemical properties analysis were conducted on the ozone-treated fruit juices samples. It was found that the survival rate of aerobic bacteria, yeast and mold in unfiltered pummelo fruit juice were higher compared to filtered juice, as the juice components acted as protective barriers to the microorganisms. The microorganisms' inactivation in pummelo fruit juices was also observed to have increased as the ozone treatment time increased. Significant effects on total colour difference, ascorbic acid content, and total phenolic content were also observed over increased ozone-treatment time. However, ozone was shown to be ineffective in activating PME activity in both types of juice. The experimental results of this study indicated that pummelo fruit juice components had significant effects on the effectiveness of gaseous ozone, however, the degree of the effects depends on the different fruit components (total soluble solids, total phenolic content). As a conclusion, filtered juice showed better quality characteristics in comparison to unfiltered juice post-ozone treatment.
Banana is the second largest cultivated fruit crop in Malaysia, and is cultivated for both the domestic market and also for export. Anthranose is a well-known postharvest disease of banana and with high potential for damaging market value, as infection commonly occurs during storage. Anthracnose symptoms were observed on several varieties of banana such as mas, berangan, awak, nangka, and rastali in the states of Perak and Penang between August and October 2011. Approximately 80% of the fruits became infected with initial symptoms characterized as brown to black spots that later became sunken lesions with orange or salmon-colored conidial masses. Infected tissues (5 × 5 mm) were surface sterilized by dipping in 1% sodium hypochlorite (NaOCl) for 3 to 5 min, rinsed with sterile distilled water, and plated onto potato dextrose agar (PDA). Direct isolation was done by transferring the conidia from conidial masses using an inoculation loop and plating onto PDA. For both methods, the PDA plates were incubated at 27 ± 1°C with cycles of 12 h light and 12 h darkness. Visible growth of mycelium was observed after 4 to 5 days of incubation. Twenty isolates with conidial masses were recovered after 7 days of incubation. The isolates produced grayish white to grayish green and grey to moss dark green colony on PDA, pale orange conidial masses, and fusiform to cylindrical and hyaline conidia with an average size of 15 to 19 × 5 to 6 μm. Appresoria were ovate to obovate, dark brown, and 9 to 15 × 7 to 12 μm and setae were present, slightly swollen at the base, with a tapered apex, and brown. The cultural and morphological characteristics of the isolates were similar to those described for C. gleosporioides (1,2,3). All the C. gloeosporioides isolates were deposited in culture collection at Plant Pathology Lab, University Sains Malaysia. For confirmation of the identity of the isolates, ITS regions were sequenced using ITS4 and ITS5 primers. The isolates were deposited in GenBank with accessions JX163228, JX163231, JX163201, JX163230, JX163215, JX163223, JX163219, JX163202, JX163225, JX163222, JX163206, JX163218, JX163208, JX163209, JX163210, JX431560, JX163212, JX163213, JX431540, and JX431562. The resulting sequences showed 99% to 100% similarity with multiple C. gloeosporioides isolates in GenBank. Pathogenicity tests were conducted using mas, berangan, awak, nangka, and rastali bananas. Fruit surfaces were sterilized with 70% ethanol and wounded using a sterile scalpel. Two inoculation techniques were performed separately: mycelia plug and conidial suspension. Mycelial disc (5 mm) and a drop of 20 μl spore suspension (106 conidia/ml) were prepared from 7-day-old culture and placed on the fruit surface. The inoculated fruits were incubated at 27 ± 1°C for 10 days at 96.1% humidity. After 3 to 4 days of inoculation, brown to black spotted lesions were observed and coalesced to become black sunken lesions. Similar anthracnose symptoms were observed on all banana varieties tested. C. gloeosporioides was reisolated from the anthracnose lesions of all the inoculated fruit in which the cultural and morphological characteristics were the same as the original isolates. To our knowledge, this is the first report of C. gloeosporioides causing anthracnose of Musa spp. in Malaysia. References: (1) P. F. Cannon et al. Mycotaxon 104:189, 2008. (2) J. E. M. Mordue. Glomerella cingulata. CMI Description of Pathogenic Fungi and Bacteria, No. 315. CAB International,1971. (3) H. Prihastuti et al. Fungal Diversity 39:89, 2009.
Hylocereus undatus widely grows in southern China. Some varieties are planted for their fruits, known as dragon fruits or Pitaya, while some varieties for their flowers known as Bawanghua. Fresh or dried flowers of Bawanghua are used as routine Chinese medicinal food. Since 2008, a serious anthracnose disease has led to great losses on Bawanghua flower production farms in the Baiyun district of Guangzhou city in China. Anthracnose symptoms on young stems of Bawanghua are reddish-brown, sunken lesions with pink masses of spores in the center. The lesions expand rapidly in the field or in storage, and may coalesce in the warm and wet environment in spring and summer in Guangzhou. Fewer flowers develop on infected stems than on healthy ones. The fungus overwinters in infected debris in the soil. The disease caused a loss of up to 50% on Bawanghua. Putative pathogenic fungi with whitish-orange colonies were isolated from a small piece of tissue (3 × 3 mm) cut from a lesion margin and cultured on potato dextrose agar in a growth chamber at 25°C, 80% RH. Dark colonies with acervuli bearing pinkish conidial masses formed 14 days later. Single celled conidia were 11 to 18 × 4 to 6 μm. Based on these morphological characteristics, the fungi were identified as Colletotrichum gloeosporioides (Penz.) Penz. & Sacc (2). To confirm this, DNA was extracted from isolate BWH1 and multilocus analyses were completed with DNA sequence data generated from partial ITS region of nrDNA, actin (ACT) and glutamine synthetase (GS) nucleotide sequences by PCR, with C. gloeosporioides specific primers as ITS4 (5'-TCCTCCGCTTATTGATATGC-3') / CgInt (5'-GGCCTCCCGCCTCCGGGCGG-3'), GS-F (5'-ATGGCCGAGTACATCTGG-3') / GS-R (5'-GAACCGTCGAAGTTCCAC-3') and actin-R (5'-ATGTGCAAGGCCGGTTTCGC-3') / actin-F (5'-TACGAGTCCTTCTGGCCCAT-3'). The sequence alignment results indicated that the obtained partial ITS sequence of 468 bp (GenBank Accession No. KF051997), actin sequence of 282 bp (KF712382), and GS sequence of 1,021 bp (KF719176) are 99%, 96%, and 95% identical to JQ676185.1 for partial ITS, FJ907430 for ACT, and FJ972589 for GS of C. gloeosporioides previously deposited, respectively. For testing its pathogenicity, 20 μl of conidia suspension (1 × 106 conidia/ml) using sterile distilled water (SDW) was inoculated into artificial wounds on six healthy young stems of Bawanghua using sterile fine-syringe needle. Meanwhile, 20 μl of SDW was inoculated on six healthy stems as a control. The inoculated stems were kept at 25°C, about 90% relative humidity. Three independent experiments were carried out. Reddish-brown lesions formed after 10 days, on 100% stems (18 in total) inoculated by C. gloeosporioides, while no lesion formed on any control. The pathogen was successfully re-isolated from the inoculated stem lesions on Bawanghua. Thus, Koch's postulates were fulfilled. Colletotrichum anthracnose has been reported on Pitaya in Japan (3), Malaysia (1) and in Brazil (4). To our knowledge, this is the first report of anthracnose disease caused by C. gloeosporioides on young stems of Bawanghua (H. undatus) in China. References: (1) M. Masyahit et al. Am. J. Appl. Sci. 6:902, 2009. (2) B. C. Sutton. Page 402 in: Colletotrichum Biology, Pathology and Control. J. A. Bailey and M. J. Jeger, eds. CAB International, Wallingford, UK, 1992. (3) S. Taba et al. Jpn. J. Phytopathol. 72:25, 2006. (4) L. M. Takahashi et al. Australas. Plant Dis. Notes 3:96, 2008.
Rambutan (Nephelium lappaceum L.) is a tropical fruit grown in Hawaii for the exotic fruit market. Fruit rot was observed periodically during 1998 and 1999 from two islands, Hawaii and Kauai, and severe fruit rot was observed during 2000 in orchards in Kurtistown and Papaikou on Hawaii. Symptoms were characterized by brown-to-black, water-soaked lesions on the fruit surface that progressed to blackening and drying of the pericarp, which often split and exposed the aril (flesh). In certain cultivars, immature, small green fruits were totally mummified. Rambutan trees with high incidence of fruit rot also showed symptoms of branch dieback and leaf spot. Lasmenia sp. Speg. sensu Sutton, identified by Centraalbureau voor Schimmelcultures (Baarn, the Netherlands), was isolated from infected fruit and necrotic leaves. Also associated with some of the fruit rot and dieback symptoms were Gliocephalotrichum simplex (J.A. Meyer) B. Wiley & E. Simmons, and G. bulbilium J.J. Ellis & Hesseltine. G. simplex was isolated from infected fruit, and G. bulbilium was isolated from discolored vascular tissues and infected fruit. Identification of species of Gliocephalotrichum was based on characteristics of conidiophores, sterile hairs, and chlamydospores (1,4). Culture characteristics were distinctive on potato dextrose agar (PDA), where the mycelium of G. bulbilium was light orange (peach) without reverse color, while G. simplex was golden-brown to grayish-yellow with dark brown reverse color. Both species produced a fruity odor after 6 days on PDA. In pathogenicity tests, healthy, washed rambutan fruits were wounded, inoculated with 30 μl of sterile distilled water (SDW) or a fungus spore suspension (105 to 106 spores per ml), and incubated in humidity chambers at room temperature (22°C) under continuous fluorescent light. Lasmenia sp. (strain KN-F99-1), G. simplex (strain KN-F2000-1), and G. bulbilium (strains KN-F2001-1 and KN-F2001-2) produced fruit rot symptoms on inoculated fruit and were reisolated from fruit with typical symptoms, fulfilling Koch's postulates. Controls (inoculated with SDW) had lower incidence or developed less severe symptoms than the fungus treatments. Inoculation tests were conducted at least twice. To our knowledge, this is the first report of Lasmenia sp. in Hawaii and the first report of the genus Gliocephalotrichum on rambutan in Hawaii. These pathogens are potentially economically important to rambutan in Hawaii. G. bulbilium has been reported previously on decaying wood of guava (Psidium guajava L.) in Hawaii (2), and the fungus causes field and postharvest rots of rambutan fruit in Thailand (3). References: (1) J. J. Ellis and C. W. Hesseltine. Bull. Torrey Bot. Club 89:21, 1962. (2) D. F. Farr et al. Fungi on Plants and Plant Products in the United States. The American Phytopathological Society, St. Paul, MN, 1989. (3) N. Visarathanonth and L. L. Ilag. Pages 51-57 in: Rambutan: Fruit Development, Postharvest Physiology and Marketing in ASEAN. ASEAN Food Handling Bureau, Kuala Lumpur, Malaysia, 1987. (4) B. J. Wiley and E. G. Simmons. Mycologia 63:575, 1971.
Citrus canker caused by Xanthomonas citri subsp. citri is a disease affecting the yield and fruit quality of lime (Citrus aurantiifolia). This research investigated endophytic bacteria obtained from six healthy Citrus spp. to inhibit the pathogen and to control citrus canker on lime plants. Numbers of the endophytic bacteria isolated from C. aurantifolia, C. hystrix, C. maxima, C. nobilis, C. reticulata and C. sinensis were 28, 25, 29, 42, 12 and 34 isolates, respectively. The selected endophytic bacteria that were effective against X. citri subsp. citri were Bacillus amyloliquefaciens LE109, B. subtilis LE24 and B. tequilensis PO80. The optimum culture medium for an antagonistic effect on the pathogen in B. amyloliquefaciens LE109 and B. tequilensis PO80 was yeast extract peptone dextrose broth, and in B. subtilis LE24 was modified soluble starch broth. To control citrus canker in lime, young expanded leaves of lime plants were aseptically punctured and inoculated with 30 μl of bacterial suspension of the pathogen (108 CFU/ml in 0.85% NaCl) per punctured location. After the pathogenic inoculation for 24 h, the leaves were then inoculated with 30 μl of the selected endophytic bacteria (108 CFU/ml in 0.85% NaCl), and treated with 30 μl of the culture media containing bioactive compounds produced by the selected endophytic bacteria. The leaves inoculated with cell suspensions of B. amyloliquefaciens LE109 or B. subtilis LE24 could completely control citrus canker. However, the leaves inoculated with B. tequilensis PO80 displayed 10% disease incidence. Additionally, the leaves treated with the crude bioactive compounds of B. amyloliquefaciens LE109 or B. subtilis LE24 could completely control citrus canker. Notably, the leaves treated with the crude bioactive compounds of B. tequilensis PO80 displayed 5% disease incidence. The results of this study showed that the Bacillus strains play important roles in the biocontrol of citrus canker in lime.