Suspended Droplets Research Articles

Gravity effect of silica and polystyrene particles on deposition pattern control and particle size distribution on hydrophobic surfaces

Ring-like residual deposits are often observed after drying suspension droplets on surfaces. That introduces a crucial defect in practical applications, such as ink-jet printing that requires a uniform deposit. In this study, depositions after drying sessile/pendant droplets with silica/polystyrene particles were examined to identify the effect of gravitational sedimentation of particles on self-pinning and deposition pattern. The suspension droplet with larger polystyrene (or silica) particles would trigger the self-pinning at larger (or smaller) particle concentrations. The deposition pattern of pendant drops with silica particles is hill-like. This is due to the large density difference between silica and water to induce high sedimentation velocity, and the particles would precipitate and aggregate along the air–liquid interface to form a hollow shell deposit. The depositions for suspensions with bi-dispersed particles, the larger silica particles would accumulate at the bottom of the deposit due to the higher sedimentation velocity, in contrast to the well-mixed in the deposits for bi-dispersed polystyrene particles. The sedimentation and capillary flow velocities were calculated and discussed. These new findings reveal that the particle density (gravity effect) can be used to adjust the sedimentation velocity to control the deposition morphology and particle size distribution even to suppress the coffee-ring effect.

Study of Contact Electrification at Liquid-Gas Interface.

It is known that the suspended liquid droplets in clouds can generate electrostatic charges, which finally results in the lightning. However, the detailed mechanism related to the contact-electrification process on the liquid-gas (L-G) interfaces is still poorly understood. Here, by introducing an acoustic levitation method for levitating a liquid droplet, we have studied the electrification mechanism at the L-G interface. The tribo-motion between water droplets and air induced by the ultrasound wave leads to the generation of positive charges on the surface of the droplets, and the charge amount of water droplets (20 μL) gradually reaches saturation within 30 s. The mixed solid particles in droplets can increase the amount of transferred charge, whereas the increase of ion concentration in the droplet can suppress the charge generation. This charge transfer phenomenon at L-G interfaces and the related analysis can be a guidance for the study in many fields, including anti-static, harvesting rainy energy, micro/nano fluidics, triboelectric power generator, surface engineering, and so on. Moreover, the surface charge generation due to L-G electrification is an inevitable effect during ultrasonic levitation, and thus, this study can also work for the applications of the ultrasonic technique.

Foliar Uptake of the Potencially Toxic Elements in Garlic Chive Leaves

Contamination of vegetables due to the foliar uptake of atmospheric toxic elements could pose severe health risks. However, the uptake mechanisms of potencially toxic elements (PTEs) from the atmosphere and translocation by plant leaves remain unclear. In this study, carboxylic acid-functionalized water-soluble CdSe/ZnS quantum dot nanoparticles (QD NPs) were used as an experimental particle model of PTEs in the edible plant garlic chive (Allium tuberosum). A droplet of QD NP suspension was deposited to simulate the conditions of raindrops containing metal particles falling on a plant leaf. The 3D spatial distribution of QD NPs in plant leaves was measured using three complementary imaging techniques: synchrotron X-ray microcomputed tomography (micro-CT), nano-CT, and two-photon microscopy (TPM). The TPM and micro-CT results revealed that QD NPs deposited on garlic chive leaves penetrated the plant leaves. Nano-CT images showed that QD NPs are absorbed into mesophyll cells and phloem vessels. The results of TEM and TPM imaging demonstrated that QD NPs penetrate through the leaves and translocate in the direction of the stem. The use of these emerging imaging techniques improved the ability to detect and visualize NPs in a plant leaf. These observations also provide mechanistic insights into foliar metal uptake and their translocation and accumulation.

Colloidal Deposits via Capillary Bridge Evaporation and Particle Sorting Thereof.

Evaporating droplets of colloidal suspensions leave behind particle deposits which could be effectively controlled via manipulating the surrounding conditions and particles and liquid properties. While previous studies extensively focused on sessile and pendant droplets, the present work investigates the evaporation dynamics of capillary bridges of colloidal suspensions formed between two parallel plates. We vary the wettability of the plates and the particle size and composition of the colloidal suspensions, keeping the same spacing between the plates. We employ side visualization, optical microscopy, fluorescence microscopy, and scanning electron microscopy and develop computational and theoretical models to collect the data. A computational model based on diffusion-limited evaporation is used to characterize the timescale of the evaporation of the capillary bridge. The model predictions are in good agreement with the present and prior experimental measurements. We discuss about the deposits of monodispersed particle suspension formed by the interplay of pinning of the contact line and evaporation dynamics. Multiple rings on the plates are observed due to the stick-slip motion of the contact line. The larger particles tend to form asymmetric deposits, with most particles concentrated on the bottom plates due to a considerably stronger gravitational pull than the hydrodynamic drag. This deposition is explained by estimating the competing forces on the particles during the evaporation. A regime map is proposed for classifying deposits on the particle size wettability plane. Lastly, we demonstrate size-based particle sorting of bidispersed colloidal suspensions in this framework. We describe two mechanisms: gravity-assisted and geometry-assisted sorting, which can be designed to sort particles efficiently. A regime map depicting the regions of influence of each mechanism is presented.

Experimental study of evaporation of droplets of water ethanol solution at high relative air humidity

A change in the temperature of suspended droplets of a water solution of ethanol, evaporating at high relative humidity, is experimentally investigated using high-speed microphotography and infrared thermography. The data obtained for ethyl alcohol droplets show a significant dependence of the surface temperature of evaporating droplets on relative humidity of the ambient air. It should be noted that at high relative humidity, a significantly smaller decrease in the temperature of droplet surface than at low humidity was observed during evaporation. This relates to the processes of moisture absorption and condensation on the surface of an evaporating droplet of ethyl alcohol at high air humidity, accompanied by the release of heat.

A Sessile Drop Method for Facile and Robust Spheroid Cultures

AbstractIn this work, an innovative sessile drop (SD) strategy for facile, robust, and long‐term cell spheroid cultures is proposed. Unlike the conventional hanging drop (HD) method, this strategy employs a superhydrophobic perforated microwell plate (SHPMP) to spatially confine arrays of cell suspension droplets for 3D spheroid cultures. Benefitting from a combination of upward positioning of cell suspension droplets and non‐wettability of the microwell surface, the SD culture platform not only maintains the technical simplicity and high oxygen availability of the HD method, but also provides high mechanical stability and ease of medium exchange, thus enabling a robust long‐term spheroid culture in a high‐throughput manner. In a proof of concept application, the convenience and simplicity of the SD approach is demonstrated by producing 384 uniform spheroids of MHCC97H cell with SHPMP. To validate the utility of this method, it is further demonstrated that spheroids fabricated by this method can be used for drug testing. With convenient liquid handling, low reagent/sample consumption, high droplet stability, adequate oxygen supply, and good in situ monitoring compatibility, the SD platform is beneficial for long‐term cell spheroid culture in a high throughput manner and provides a powerful tool for tissue engineering, regenerative medicine, and drug screening.

Evaluation of Microfluidic Approaches to Encapsulate Cells into PEGDA Microparticles

PurposePolyethylene glycol diacrylate (PEGDA) is increasingly used to microencapsulate cells via a vortex-induced water-in-oil emulsion process. Herein, we evaluated methods to encapsulate cells into microparticles using microfluidic methods.MethodsPEGDA prepolymer solution with or without cells was photopolymerized with white light under varying microfluidic parameters to form empty microspheres or cell-laden microparticles. Microparticles and entrapped cells were assessed for size and viability.ResultsPEGDA microparticles were easily formed when cells were absent; the introduction of cells resulted in aggregation that clogged microfluidic devices, resulting in a mix of empty polymer microparticles and cells that were not encapsulated. Cells that were successfully encapsulated had poor viability.ConclusionMicrofluidic methods may work for low density microencapsulation of mammalian cells; however, when the cell density within each microparticle must be relatively high, emulsion-based methods are superior to microfluidic methods.Lay SummaryThe synthetic polymer polyethylene glycol diacrylate (PEGDA) has been increasingly used to encapsulate cells into micrometer-sized hydrogel spheres (microspheres). One method to microencapsulate cells has been to form a water-in-oil emulsion with liquid polymer containing cells and then expose the suspended droplets to white light, polymerizing them into PEGDA hydrogel microspheres. Although successful, this method has poor control over the process, resulting in polydisperse microsphere sizes with varying cell density. We evaluated microfluidic methods to form both empty and cell-laden PEGDA microspheres. Although microfluidic methods resulted in monodisperse microsphere sizes, the introduction of cells resulted in clogging of microfluidic devices, non-spherical microparticles, and poor cell viability.Future WorkBecause the microfluidic approach successfully formed cell-free microspheres, the effect of reducing cell aggregation will be examined. Specifically, the use of anti-aggregation agents as well as a reduced cell density in the liquid polymer phase and their effects on polymer formation will be explored.

Regulation of biomolecular condensates by interfacial protein clusters.

Biomolecular condensates are cellular compartments that can form by phase separation in the absence of limiting membranes. Studying the P granules of Caenorhabditis elegans, we find that condensate dynamics are regulated by protein clusters that adsorb to the condensate interface. Using in vitro reconstitution, live observations, and theory, we demonstrate that localized assembly of P granules is controlled by MEG-3, an intrinsically disordered protein that forms low dynamic assemblies on P granules. Following classic Pickering emulsion theory, MEG-3 clusters lower surface tension and slow down coarsening. During zygote polarization, MEG-3 recruits the DYRK family kinase MBK-2 to accelerate spatially regulated growth of the P granule emulsion. By tuning condensate-cytoplasm exchange, interfacial clusters regulate the structural integrity of biomolecular condensates, reminiscent of the role of lipid bilayers in membrane-bound organelles.

A new conceptual model for adiabatic fog

Abstract. Visibility reduction caused by fog can be hazardous for human activities, especially for the transport sector. Previous studies show that this problem could be mitigated by improving nowcasting of fog dissipation. To address this issue, we propose a new paradigm which could potentially improve our understanding of the life cycle of adiabatic continental fogs and of the conditions that must take place for fog dissipation. For this purpose, adiabatic fog is defined as a layer filled with suspended liquid water droplets, extending from an upper boundary all the way down to the surface, with a saturated adiabatic temperature profile. In this layer, the liquid water path (LWP) must exceed a critical value: the critical liquid water path (CLWP). When the LWP is less than the CLWP, the amount of fog liquid water is not sufficient to extend all the way down to the surface, leading to a surface horizontal visibility greater than 1 km. Conversely, when the LWP exceeds the CLWP, the amount of cloud water is enough to reach the surface, inducing a horizontal visibility of less than 1 km. The excess water with respect to the critical value is defined as the reservoir liquid water path (RLWP). The new fog paradigm is formulated as a conceptual model that relates the liquid water path of adiabatic fog with its thickness and surface liquid water content and allows the critical and reservoir liquid water paths to be computed. Both variables can be tracked in real time using vertical profiling measurements, enabling a real-time diagnostic of fog status. The conceptual model is tested using data from 7 years of measurements performed at the SIRTA observatory, combining cloud radar, microwave radiometer, ceilometer, scatterometer, and weather station measurements. In this time period we found 80 fog events with reliable measurements, with 56 of these lasting more than 3 h. The paper presents the conceptual model and its capability to derive the LWP from the fog top height and surface horizontal visibility with an uncertainty of 10.5 g m−2. The impact of fog liquid water path and fog top height variations on fog life cycle (formation to dissipation) is presented based on four case studies and statistics derived from 56 fog events. Our results, based on measurements and an empirical parametrization for the adiabaticity, validate the applicability of the model. The calculated reservoir liquid water path is consistently positive during the mature phase of fog and starts to decrease quasi-monotonously about 1 h before dissipation, reaching a near-zero value at the time of dissipation. Hence, the reservoir liquid water path and its time derivative could be used as indicators of the life cycle stage, to support nowcasting of fog dissipation.

Secondary breakup of shear thickening suspension drop

To explore the effect of shear thickening behavior on the secondary deformation and breakup of cornstarch–water suspension droplets, an experimental investigation is conducted by using a high-speed camera. The experimental results demonstrate suspension droplets that exhibit discontinuous shear thickening (DST) exhibit a hardened deformation mode when they fall into the airflow field. When the droplets are in a hardened deformation mode, the windward side of the droplet deforms into a sheet, while the leeward side remains hemispherical until the droplet leaves the airflow field. The dimensionless number N is established to describe the relative magnitude of the increment of the viscous force and aerodynamic force during the secondary breakup process. Based on the suggested dimensionless number N and the Weber number We, the secondary deformation and breakup regime map of Newtonian fluids and DST suspensions is also proposed.

First Report of Colletotrichum aenigma Causing Anthracnose of Grape in Korea.

Grape (cv. Kyoho) is one of the most popular dessert fruits in South Korea. Anthracnose caused by Colletotrichum species is a common and very destructive disease of grape in the country. In 2019, severe outbreaks of anthracnose was observed in different grape orchards in Gimcheon (36º09´N, 128º00´ E), South Korea. The disease incidence on fruit was up to 50% in the orchards with most severe outbreaks and infected fruit displayed typical anthracnose symptoms including sunken necrotic lesions with orange-like conidial mass. For isolation of putative causal agents, nine diseased fruits were collected from three commercial orchards. A total of nineisolates were made from nine of the infected fruit by spreading spore masses (1x106 conidia mL-1) from each fruit on water agar and collecting single germinated spores after incubation at 25 ºC overnigh. The single germinated spores were transferred on to fresh potato dextrose agar (PDA) (Difco, Becton Dickinson) and incubated at 25ºC in the dark. Seven day old colonies were cottony white on the upper side and gray at the center on the reverse side. Conidia were cylindrical with round ends and measured 13.9 - 20.1 × 5.4 - 8.1 μm (mean = 16.5 × 6.6 μm, n = 30). Appressoria were brownish, sub-cylindrical with a few lobes and 10.3 -16.7 × 6.6 - 10.9 μm (mean = 13.1 × 8.1 μm, n = 30). The morphological characteristics of the solates resembled those of Colletotrichum species within the C. gloeosporioides complex (Weir et al. 2012). DNA was amplified using the following primer pairs: ITS1/ITS4, GDF / GDR, ACT-512F / ACT-783R, Bt2a/ Bt2b, and CHS79-F/CHS-354R (Weir et al. 2012). Accession numbers, LC586811 to LC586825 were obtained after depositing all the resulting sequences in GenBank. A 50% majority rules phylogenetic tree (Bayesian phylogenic analysis) was constructed based on concatenated sequences of ITS, GAPDH, ACT, TUB, and CHS using MrBayes 3.2.10. The present isolates formed a single clade with the reference isolates of C. aenigma (isolate ICMP 18608 and ICMP 18686). For a pathogenicity test, healthy grapefruits were collected from an orchards, surface sterilized by dipping in 1% sodium hypochlorite, rinsed with sterilized water and dried by blotting. A conidial suspension (1×106 conidia mL-1) in sterilized water were prepared from one week old colonies of isolates GRAP10 and GRAP12. A small wound was made on sterilized detached fruit by punching with a sterile pin. A drop of the conidial suspension was placed on the wound, while the control fruit received a drop of sterile water. Similarly, unwounded fruit were also inoculated with a single droplet of conidial suspension. For each isolate and method (wounded and unwounded), ten fruit were inoculated, and ten non-inoculated fruit were used as control. All the treated fruit were kept in a plastic box containing moist tissue and incubated at 25º C in the dark. Typical anthracnose lesions appeared on all inoculated wounded fruit while non-inoculated and inoculated unwounded fruits remained asymptotic. Koch postulates were fulfilled by re-isolating and re-identifying the causal agent from inoculated fruit. Colletotrichum aenigma has been reported as the causal agent of anthracnose on Juglans regia, Camellia sinensis and Actinidia arguta in China (Weir et al. 2012; Wang et al. 2016; Wang et al. 2018). Previous studies reported four Colletotrichum species (C. acutatum, C. gloeosporioides, C. fructicola, and C. viniferum) to cause this disease on grapes in South Korea (Oo and Oh 2017; Lim et al. 2020). To the best of our knowledge, this is the first report on grape anthracnose caused by C. aenigma in South Korea. This finding may help to take effective control measures of this disease.

Experimental study on the evaporation performance of suspended CeO2 nano-fuel droplets

Experimental study on the evaporation performance of suspended CeO2 nano-fuel droplets

Photocatalytic Treatments for Personal Protective Equipment: Experimental Microbiological Investigations and Perspectives for the Enhancement of Antimicrobial Activity by Micrometric TiO2.

The COVID-19 pandemic has led to countries enforcing the use of facial masks to prevent contagion. However, acquisition, reuse, and disposal of personal protective equipment (PPE) has generated problems, in regard to the safety of individuals and environmental sustainability. Effective strategies to reprocess and disinfect PPE are needed to improve the efficacy and durability of this equipment and to reduce waste load. Thus, the addition of photocatalytic materials to these materials, combined with light exposure at specific wavelengths, may represent promising solutions. To this aim, we prepared a series of masks by depositing micrometer-sized TiO2 on the external surfaces; the masks were then contaminated with droplets of bacteria suspensions and the coatings were activated by light radiation at different wavelengths. A significant reduction in the microbial load (over 90%, p < 0.01) was observed using both Gram negative (E. coli) and Gram positive (S. aureus) bacteria within 15 min of irradiation, with UV or visible light, including sunlight or artificial sources. Our results support the need for further investigations on self-disinfecting masks and other disposable PPE, which could positively impact (i) the safety of operators/workers, and (ii) environmental sustainability in different occupational or recreational settings.

Evaporation and combustion characteristics for a droplet of lubricating oil and gasoline

The droplet suspension technology is used to study the effects of ambient temperatures (from 673 to 873 K) and gasoline blending ratios (from 0% to 20%) on evaporation and combustion characteristics of lubricating oil (LO), used lubricating oil (ULO) and the mixture of lubricating oil and gasoline (LO/gasoline and ULO/gasoline). The experimental results show that the evaporation process of LO80G20 (the blend of 80% LO and 20% gasoline by volume fraction) contains a transient heating period, a fluctuation evaporation period and a slow evaporation period at 673 K. The combustion process at high temperatures (from 723 to 873 K) includes an auto-ignition delay period, a combustion period and an extinction period. At the same ambient temperature, both the auto-ignition delay and lifetime of ULO are shorter than those of new LO. The occurrence time and intensity of puffing for ULO are earlier and stronger than those of new LO. For the mixture of lubricating oil and gasoline (LO/gasoline and ULO/gasoline), the auto-ignition delay, droplet lifetime and the occurrence time of puffing decrease with the increase of gasoline component.

A superhydrophobic chip integrated with an array of medium reservoirs for long-term hanging drop spheroid culture

Hanging drop (HD) is one of the most popular methods used for forming three-dimensional (3D) cell spheroids. However, conventional hanging drop systems are only applicable for short-term spheroid culture due to their inconvenience in exchanging cell culture media. Here we present a medium-reservoir-integrated superhydrophobic (MRI-SH) chip for long-term HD spheroid cultures. The device consists of two main components: i) a patterned superhydrophobic (SH) surface containing an array of wettable spots which anchor arrays of droplets of cell suspension, and ii) an array of chambers that serve as medium reservoirs, both interconnected via an array of thru-holes. This configuration provides two distinct advantages over conventional HD configurations: i) the high wettability contrast of the SH pattern on the chip leads to the formation and adhesion of nearly spherical hanging droplets on its surface, which minimizes interactions between the liquid and the substrate; ii) the integrated chambers provide large volumes of medium to maintain longer culture durations. Using this device, spheroids of MHCC97H cells were successfully formed, and the cultured spheroids could maintain high viability for up to 30 days and exhibited enhanced spheroid morphology compared to those cultured in the conventional HD systems. Statement of significanceThis paper presents a medium-reservoir-integrated superhydrophobic hanging drop (HD) platform for the long-term culture of spheroids with enhanced morphology. By monolithically integrating medium reservoirs and a patterned SH surface into a single device, this HD platform can not only produce high-quality spheroids, but also permit them to sustain high viability for up to 30 days without the need for tedious medium replenishment. We believe that such a platform will be valuable in a wide range of biological or biomedical applications, including tissue engineering, regenerative medicine, and drug discovery.

Scientific Evaluation of Dhuma Yoga (An Ayurvedic Fumigation Medicine) as Preventive and Curative on Symptoms of Covid-19 Disease

Covid-19, an infectious disease caused by a novel coronavirus SARS-CoV-2 spreads primarily through droplets of saliva or discharge from the nose when an infected person talks, coughs or sneezes where the viruses are active in the environment within the suspended micro droplets. Sanitization of environment to weaken/terminate the virus and halting the replication of virus inside the host along with symptomatic treatment is the primary approach to end the pandemic. In Ayurveda, Dhupana (medicated fumigation of vicinity) and Dhumapana (medicated smoking) therapies done by drugs of herbal/animal/mineral origin are a swift way to decontaminate the environment and Respiratory system. Dhuma (medicated fumes) is a unique drug delivery system acting directly on respiratory tissues which can deliver quick results in this Covid-19 pandemic by its local and systematic effects recommended by AYUSH ministry in the guidelines for Covid-19. We intend to put forward the scientific explanation of powerful Ayurvedic Cannabis based polyherbal dhumapana (medicated smoking) medication named Dhuma Yoga available in the market as an alternate remedy for Covid-19 whose four out of five herbal ingredients are in the list of WHO manual of traditional medicine. We emphasized on Vijaya (Cannabis sativa Linn.) as the centre of formulation because it is a Rasayana (rejuvenative) herb having Vyavayi (fast acting) and Yogavahi (synergetic) properties. Phytochemicals of all the herbal ingredients of Dhuma Yoga formulation are studied through in silico, In vitro and In vivo studies for Covid-19 with favourable outcomes.

Determination of the Toxic Potential of the Formation of Oil-Suspended Particulate Matter Aggregates (OSA) in a Simulated Microscale Experiment

The aggregation of suspended particulate matter (SPM) and oil droplets in environments with specific hydrodynamic energy can lead to the formation of Oil-SPM Aggregates (OSA). A laboratory simulation was conducted in order to examine the possible toxic potential of OSA formation using three different concentrations of particulate material (50, 200, 300 mg/L) in a microscale experiment. The procedure was performed through toxicological testing of acute exposure to determine the LC 50 (lethal concentration 50%) using the microcrustacean Artemia salina as a test organism. Serial dilutions were made from surface and bottom samples in order to characterize different toxicity. The concentration that showed the highest potential toxicity was 200 mg/L, having the same values for surface and bottom (LC 50 7.91%), whereas the concentration with the least toxic potential was 300 mg/L (LC 50 31.5%) for surface samples. Negative correlation was found between redox potential and the hydrogenionic potential (only for samples with 200 mg/L of sediment), and positive correlation was found between toxicity factors (percent dilution and mortality) and the other monitored parameters.

Real-time holographic quantitative measurement of vapor density distribution of suspended droplets.

We applied digital holography (DH) technology in a quantitative measurement of the density distribution of a low refractive index transparent substance (e.g., the vapor of suspended droplets). An optical setup was built based on the Mach-Zehnder interferometer. A measurement performance test showed the mean relative error of the measurement error was about 2.0%; that of the environment disturbance error was about 0.47%. By a quantitative method to assess the precision limit, the temperature measurement precision could achieve 0.01°C, and the vapor density measurement precision could achieve 0.0001kg/m3. We believe that all the benefits above make the setup a good choice for application in the Chinese space station.

Water-in-Oil Nano-Emulsions Prepared by Spontaneous Emulsification: New Insights on the Formulation Process.

Nano-emulsions consist of stable suspensions of nano-scaled droplets that have huge loading capacities and are formulated with safe compounds. For these reasons, a large number of studies have described the potential uses of nano-emulsions, focusing on various aspects such as formulation processes, loading capabilities, and surface modifications. These studies typically concern direct nano-emulsions (i.e., oil-in-water), whereas studies on reverse nano-emulsions (i.e., water-in-oil) remain anecdotal. However, reverse nano-emulsion technology is very promising (e.g., as an alternative to liposome technology) for the development of drug delivery systems that encapsulate hydrophilic compounds within double droplets. The spontaneous emulsification process has the added advantages of optimization of the energetic yield, potential for industrial scale-up, improved loading capabilities, and preservation of fragile compounds targeted for encapsulation. In this study, we propose a detailed investigation of the processes and formulation parameters involved in the spontaneous nano-emulsification that produces water-in-oil nano-emulsions. The following details were addressed: (i) the order of mixing of the different compounds (method A and method B), (ii) mixing rates, (iii) amount of surfactants, (iv) type and mixture of surfactants, (v) amount of dispersed phase, and (vi) influence of the nature of the oil. The results emphasized the effects of the formulation parameters (e.g., the volume fraction of the dispersed phase, nature or concentration of surfactant, or nature of the oil) on the nature and properties of the nano-emulsions formed.

First Report of Leaf Spot Caused by Colletotrichum fructicola and C. siamense on Zizyphus mauritiana in Guangxi, China.

Zizyphus mauritiana Lam. is an important tropical fruit tree and has significant economic value. It is widely planted in Hainan, Guangdong, Guangxi and Fujian provinces in China (Yang et al. 2017). In March 2019, leaf spot was observed on leaves of Z. mauritiana at Bagui fields in Nanning, Guangxi, China, with incidence exceeding 50%. Symptomatic leaves developed a yellow to tan-brown sunken lesion and finally abscised. To isolate the pathogen causing the symptoms, small pieces (5 × 5 mm) of infected leaves were surface sterilized by exposure to 75% ethanol for 10 sec, 1% sodium hypochlorite for 1 min and rinsed three times in sterile water. Fifty pieces were isolated, surface sterilized, and pieces were plated onto potato dextrose agar (PDA) and grown at 28°C for 7 days. The isolation rate of Colletotrichum species was 100%. Three representative isolates (DQZ3-1, DQZ3-2 and DQZ3-3) were selected for further study. Mycelia were greyish-white for all three isolates, with isolate DQZ3-1 also appearing dark green in the center of the colony. Conidia were elliptical, aseptate and hyaline, with sizes of 13.4 ± 0.12 µm × 5.7 ± 0.1 µm, 14.8 ± 0.1 µm × 5.8 ± 0.1 µm and 15.1 ± 0.1 µm × 5.5 ± 0.1 µm for DQZ3-1, DQZ3-2 and DQZ3-3, respectively. Genomic DNA was extracted using the DNAsecure Plant Kit [Tiangen Biotech (Beijing) Co., Ltd] and the internal transcribed spacer (ITS), partial actin (ACT), calmodulin (CAL), chitin synthase (CHS-1), beta-tubulin (TUB2), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were sequenced (Weir et al. 2012). Phylogenetic analysis of the three isolates was performed with MEGA-X (Version 10.0) based on sequences of multiple loci (ITS, ACT, CAL, CHS-1, TUB2 and GAPDH) using Maximum Likelihood analysis. Isolate DQZ3-1 was identified as C. fructicola, and the other two isolates, DQZ3-2 and DQZ3-3, were identified as C. siamense (accessions MT039396 to MT039410, for ACT, CAL, CHS-1, GAPDH and TUB2 of DQZ3-1, DQZ3-2 and DQZ3-3; MT041651 to MT041653 for ITS of DQZ3-1, DQZ3-2 and DQZ3-3). Pathogenicity tests were conducted on 1-year-old plants. Young, healthy leaves were artificially wounded by gently scratching with a sterile needle and 10 µl droplets of conidial suspension (106 spores/ml) applied per wound site for each isolate. Some wounded leaves were inoculated with 10 µl droplets of water as controls. Each isolate was inoculated onto three plants, with 15 leaves at least for each plant, same as controls. All inoculated plants were sprayed with water and covered with plastic bags to maintain high humidity. Symptomatic lesions were observed on the inoculated leaves after 7 days at 28°C, whereas no symptoms were observed on the control leaves. To fulfill Koch's postulates, fungi were re-isolated from 50 symptomatic leaf pieces and fungi re-isolated from each leaf piece were morphologically identical to the inoculated isolates, for a 100% isolation frequency. To our knowledge, this is the first report of leaf spot caused by C. fructicola and C. siamense on Z. mauritiana worldwide. This research may accelerate the development of future epidemiological studies and management strategies for anthracnose caused by C. fructicola and C. siamense on Z. mauritiana.