First report of root rot on Manglietia decidua caused by Calonectria ilicicola in China

Manglietia decidua (Magnoliaceae) was a class I endangered plant in China. During 2018-2020, a severe root rot (about 10% - 90% disease incidence ) was observed on 2-year-old seedlings in the nursery in Yichun, Jiangxi province (N27°52’20”; E114°27’46”). Symptoms started on leaves showing dehydration and chlorosis, the root of diseased plant became black and rotted, and in severe cases, the plants withered and died. The symptomatic root tissues were cut and dipped in a 3% hydrogen peroxide solution for 5 mins, rinsed thrice with sterile water, and then placed on potato glucose agar medium containing ampicillin (50mg/L). The plates were kept in an incubator at 25-28°C for 2-3 days in the dark. The Calonectria-like fungus was consistently isolated from 100% of tissues and the colonies were feathery, moderate white aerial mycelium, surface pale brown, reverse with white outer margin, and brown inner region. The perithecia produced on carnation leaf agar were solitary, subglobose to ovoid, dark red-brown, and measured 273.8 - 427.2 × 362.6 - 628.9 µm (av. 360.9 × 429.9 µm) (n = 31). Clavate asci contained eight spores and tapered into a long thin stalk. Ascospores were hyaline, guttulate, straight to slightly curved with rounded ends, 36.8- 66.1×4.4-7.3 μm (av. 49.9 × 5.9 μm) (n = 52), 1-septate, constricted at the septum and aggregated in the upper third of the ascus. On PDA，conidia formed on penicillate conidiophores within 10 days were hyaline, 1(-3)-septate, cylindrical, rounded at both ends, straight, 36.5-61.7 × 5.0-7.2 μm (av. 50.7 × 6.2 µm) (n=48). Isolate HML 20 and 27 were used to further confirm species identity by five loci analysis：ITS (MZ389092 and MZ389093), ACT (MZ398252 and MZ398253), HIS3 (MZ398254 and MZ398255), TEF1-α (MZ398256 and MZ398257), and TUB2 (MZ398258 and MZ398259). NCBI BLASTN showed the high sequence identity with Calonectria ilicicola ex-type culture CBS 190.50 (CMW 30998) (Liu et al 2020): 100 % for ITS (MT359727), TUB2 (AY725631), and HIS3 (MT335506), 99.22% for ACT (MT335036), 99.80% for TEF1-α (MT412797). Maximum likelihood (ML) analysis and Bayesian inference (BI) based on the combined ITS, tub2, his3 and tef1 sequence using RAxML v.1.0.0 and MrBayes v. 3.2.1 software revealed that isolate HML 20 and 27 clustered together with C. ilicicola strains in C. kyotensis species complex. Thus, the fungus was identified as C. ilicicola (anamorph: Cylindrocladium parasiticum) based on morpho-molecular criteria (Lombard et al. 2010). Pathogenicity was determined under greenhouse conditions (25-30 ℃). The 2-year-old plants grown in 25-cm pots for 20 days were inoculated. Five 6-mm mycelial plugs from 7-day culture on PDA were buried 5 cm under the soil adjacent to the unwounded taproot of each plant and the plants were watered regularly to keep the soil moisture content at about 15%. After ten days, inoculated plants began to show chlorosis symptoms on leaves and collapsed within 15 to 20 days, while no symptoms were observed on control plants. The same colonial fungus was successfully reisolated. Calonectria ilicicola is an economically important plant pathogen worldwide, which causes diseases on Arachis hypogaea, Cinnamomum kanahirai, Glycine max, Medicago sativa, Sassafras randaiense, and Vaccinium spp. etc. in China (Gai et al 2017, Fei et al 2018, Zhang et al 2020 ). As far as we know, it is first report of C. ilicicola causing root rot on M. decidua. At present, this disease is an important threat to the conservation of M. decidua.

Detection of INVA Gene and Cytotoxin of Salmonella entertidis in Food Samples Using Molecular Methods

Salmonella entertidis is a foodborne pathogen that causes various diseases in human beings worldwide. The toxin of Salmonella can cause infectious diseases. In this research project, Salmonella was detected through various microbial, biochemical and molecular tests in diverse food samples collected from highly populated, moderately populated and less populated areas of Lahore, Pakistan. Enriched cultures of all food samples such as apples, tomatoes, yogurt and mayonnaise was streaked on violet-red bile glucose agar, Simmon’s citrate agar and eosin-methylene blue agar (EMB). Salmonella isolates were screened for the presence of toxin encoding gene through PCR. 27% apples, 19% tomatoes, 5% mayonnaise and 7% yogurt were found to be positive for INVA genes (invasion protein genes). In medical and pharmaceutical point of views the INVA gene can also help to develop specific medicines against salmonella. The cytotoxin that is protein in nature was confirmed by SDS PAGE in mayonnaise samples. This study illustrates that foods of highly populated areas are reservoir for Salmonella entertidis in Pakistan. There is need to develop specific drugs, precautionary measures to control salmonella and its disease.

Genetic diversity and antifungal susceptibility of Candida albicans isolates from Iranian HIV-infected patients with oral candidiasis

Objective The objectives of this study were to investigate the antifungal susceptibility and genetic diversity of Candida albicans isolated from HIV+ patients with oropharyngeal candidiasis. A total of 50 C. albicans isolates were cultured on Sabouraud glucose agar containing chloramophenicol. The antifungal susceptibility of the isolates against fluconazole, clotrimazole, nystatin, amphotericin B, ketoconazole and flucytosine was assessed using disc diffusion method. The genetic diversity of C. albicans isolates was determined using random amplified polymorphic DNA marker. Results The inhibition zones ranged from 4 ± 1.8 to 40 ± 3.8 mm for fluconazole, 7 ± 1.0 to 37 ± 1.8 mm for ketoconazole, 14 ± 0.8 to24 ± 0.8 mm for amphotericin B, 25 ± 0.0 to 33 ± 0.0 mm for nystatin and 7 ± 4.2 to 40 ± 0.0 mm for clotrimazole. At 90% similarity, three distinct groups were observed. The smallest cluster composed of 3 isolates, whereas the largest one composed of 17 isolates. 32% (16/50), 28% (14/50) and 14% (7/50) were resistant to fluconazole, ketoconazole and clotrimazole, respectively.

Genetic Diversity and Antifungal Susceptibility of Candida Albicans Strains Isolated from Lranian HIV+ Patients With Oral Candidiasis

Objective: The objectives of this study were to investigate the antifungal susceptibility and genetic diversity of oral Candida albicans strains isolated from HIV+ patients with oropharyngeal candidiasis. A total of 50 C. albicans isolates were cultured on Sabouraud glucose agar containing chloramohenicol. The antifungal susceptibility of C. albicans against fluconazole, clotrimazole, nystatin, amphotericin B, ketoconazole and flucytosine was assessed using disc diffusion method. The genetic diversity of different C. albicans strains was determined using random amplified polymorphic DNA technique. Results: The inhibition zones ranged from 4±1.8 to 40±3.8 mm for fluconazole, 7±1.0 to 37±1.8 mm for ketoconazole, 14±0.8 to24±0.8 mm for amphotericin B, 25±0.0 to 33±0.0 mm for nystatin and 7±4.2 to 40±0.0 mm for clotrimazole. At 90% similarity, three distinct groups were observed. The smallest cluster composed of 3 of 50 C. albicans isolates, whereas the largest cluster composed of 17 of 50 isolates. Of 50 C. albicans isolates, 32%, 28% and 14% were resistant to fluconazole, ketoconazole and clotrimazole, respectively. There were no significant differences among antifungal susceptibility of different C. albicans strains from three genotype clusters.

CHARACTERIZATION OF SYMBIOTIC EFFECTIVENESS OF RHIZOBIA NODULATING FIELD PEA (PISUM SATIVUM).

Field Pea is one of the most important Legumes plants and widely grown in Ethiopia. A study was made to re- isolate, characterize, and select best rhizobia for field pea. Results showed that all the 25 isolates exhibited typical colony characteristics and presumptive reactions of fast growing rhizobia. Out of the 25 isolates, 3(KL3, BR1 andCF5) relatively superior isolates were selected in sterilized sand. All isolates characterized their morphological and physiological characteristics. All isolates formed watery and mucoid colonies on YEMA medium, their mean growth time mostly between 2 &4 hours and failed to grow on peptone glucose agar medium and to solubilize inorganic phosphate. Almost all isolates were tolerated to pH 5to 9, 2% and 3% salt concentration, and at temperature of 15oC to 35oC. The isolates were also tolerant to erythromycin, streptomycin and ampicillin, and relatively sensitive to penicillin and chloroamphenicol at concentration of 50μg/ml. All isolates utilized to sucrose, glucanate, galactose and fructose as the sole source of carbon, and almost all isolates grow on YEMA medium containing galactose (90%), fructose (88.9%) and glucanate (76.7%) and the isolates utilized many amino acids as the source of nitrogen. BR1 was the most competitive inoculant with nodule occupancy of 75%; followed by KL3 and CF5 with nodule occupancy of 60 and 50% respectively. The mean nodule number, nodule dry weight, mean shoot dry weight and N content and of the host plants inoculated with different isolates showed variations. Particularly BR1 can be recommended as inoculants and good strain for field pea in the future.

CHARACTERIZATION OF EGGPLANT ENDOPHYTE BACTERIA AND RHIZOBACTERIA AS WELL AS THEIR ANTAGONISTIC ABILITY AGAINST Ralstonia solanacearum

Characterization of eggplant endophyte bacteria and rhizobacteria as well as their antagonistic ability against Ralstonia solanacearum. Bacterial wilt caused by Ralsonia solanacearum is one of important diseases causing severe loses in eggplant production. Various strategies were used to manage bacterial wilt, including planting resistant varieties, soil amandement, and soil solarization. However, management of R. solanacearum in eggplant by using endophytic bacteria and rhizobacteria were not been done that much. The objective of this study was to: (1) characterization of endophytic and rhizobacteria; (2) determines the inhibition ability of endophytic and rhizobacteria isolates against R. solanacearum pathogen on eggplant. The laboratory experiment was arranged in completely randomized design with 5 treatments and 5 replications. The double layer method using yeast peptone glucose agar (YPGA) medium was used in vitro test. Based on the morphological characteristics these isolates were suspected as a member of genus Bacillus. Among the isolates used in this study, TK isolate showed the best capability to inhibit growth of R. solanacearum.

Survey on the Presence of Malassezia spp. in Healthy Rabbit Ear Canals

Malassezia spp. have rarely been reported in rodents and lagomorphs. In 2011, Malassezia cuniculi was described in two rabbits. Further microscopic studies showed M. cuniculi-like yeasts in more than 50% of samples from rabbits’ ear canals, but no isolation was made. The present study details the presence of Malassezia spp. and tries to typify it from ear canals of healthy rabbits. Seventy-eight half-breed rabbits from rural farms and 98 companion dwarf rabbits from northern Italy were considered. A first attempt to screen ear swabs was performed by microscopic and cultural examination on Sabouraud Glucose Agar (SGA), modified Dixon Agar (mDA) and Leeming and Notman Agar (LNA). Additionally, ear swabs from eight further microscopically positive rabbits for M. cuniculi-like cells, were used for both isolation on LNA medium and nine of its variants and for DNA extraction, PCR and sequencing. The microscopic observation of the swabs of the screened 168 rabbits highlighted the presence of yeasts in one or both of the external ear canals of 98 rabbits (58.3%). Rabbits used for meat production were more frequently diagnosed positive than pet rabbits (P = 0.001), and young ones were more often positive compared to rabbits older than 3 months (P = 0.027). No yeast growth was observed in culture. From the eight selected rabbits, Malassezia isolation failed both on LNA and on the modified mediums. Sequences of ~300 bp fragments of 18s rDNA, obtained by PCR from swabs, showed 99.9% identity with Malassezia phylotype 131 described from human ear canals. As Malassezia-like yeasts have been observed in more than half of the examined population, its colonization of ear meatus can be considered as physiological in rabbits. The results outline how much remains to be discovered on Malassezia as a component of the skin mycobiota of rabbits and that the use of the culture examination alone is not the best choice to detect Malassezia-like yeasts in rabbits.

Exo-Inulinase Production by a Catabolite Repression-Resistant Mutant Thermophilic Aspergillus tamarii-U4 in Solid State Fermentation

In this study, spores of inulinase-producing thermophilic Aspergillus tamarii were subjected to UV mutagenesis, and colonies obtained were screened for inulinase production on inulin-glucose agar. The thermal stability of the inulinase was also investigated. A mutant strain U4 was found to produce 2.8 times inulinase titre (62.1U/mL) as against the wild strain (22.2U/mL). Inulinase production by this U4 strain was also found not to be significantly (P≤0.05) affected by the presence of glucose. The inulinase produced retained 64% of its activity after incubation at 65ºC for three hours. Solid-state fermentation for inulinase production by the strain U4 showed that wheat bran supported the highest inulinase titre 218.3U/gds while banana peels supported the lowest inulinase production titre of 80.5U/gds. Further optimization of cultural parameters revealed that incubation time of 5 days, 60% initial moisture content of the substrate, 2% inoculum density 2%, temperature 55ºC and pH 4.5 were optimal for inulinase production. Under optimized conditions, inulinase titre of 426.6 U/gds was observed. The pattern of inulin hydrolysis by the inulinase revealed the presence of monosaccharide as the main product of hydrolysis. Inulinase production at elevated temperatures by the mutant Aspergillus tamarii-U4 and its catabolite resistant properties showed that the organism is a potential industrial candidate for the production of exo-acting inulinases.