Spore Suspension Research Articles

Beneficial and biocontrol effects of Trichoderma atroviride, a dominant species in white birch rhizosphere soil.

White birch (Betula platyphylla Suk.) is a typical pioneer tree species that is important in forest restoration in northern China, Japan, and Korea. In the present study, 37 isolates were obtained from B. platyphylla rhizosphere soils in Heilongjiang Province; they were identified as T. pleuroticola (3 isolates), T. virens (2 isolates), T. hamatum (8 isolates), T. atroviride (21 isolates, dominant species) and T. asperelloides (3 isolates). Stress tolerance tests (salt, alkali, and nutritional stress that simulated saline alkali or barren soil) and confrontation assays (with four pathogens) were performed to determine which isolates had good biocontrol ability in barren soil; the results show that T. atroviride was outstanding. Then, in order to determine the effect of T. atroviride on plants and soil, Gynura cusimbua seeds were sown and treated with a T. atroviride spore suspension, as was unsown soil. The seedlings treated using T. atroviride had significantly greater height, stem diameter, soluble protein content, soluble sugar content, and malonaldehyde (MDA) content and their catalase (CAT) activity was also significantly increased. In addition, when the plants were inoculated with Alternaria alternata, the plants treated using T. atroviride had stronger CAT activity, significantly higher soluble protein content and soluble sugar content, and significantly lower MDA content, which indicates stronger resistance and less injury caused by the pathogen. In addition, T. atroviride not only increased the content of available nitrogen and available phosphorus in the soil, but also promoted G. cusimbua seedlings' absorption of available nitrogen and available phosphorus. Thus, the characteristics of T. atroviride may make it the main factor that helps B. platyphylla colonise cut-over lands. T. atroviride, a promising biocontrol candidate, can be used in agriculture and forestry.

First report of Cephalerous virescens causing Algal leaf spot of Averrhoa carambola in India.

Averrhoa carambola (Star fruit) is a drought resistant edible fruit belongs to family Oxalidaceae. It is native of Malaysia and further cultivation is extended to China, Southeast Asia, India and Northern South America. Star fruit has juicy texture and used in salads, beverages and traditionally it has been used for ayurvedic medicines in India, Brazil and China (Abduh et al. 2023). In early January 2023, we observed the symptoms of raised, more or less circular, orange to dark brown, velvet textured, scattered algal leaf spots (1-4 mm) on the upper surface of A. carambola leaves at College farm, Agricultural College, Aswaraopet (17.252039 latitude, 81.109573 longitude) (Supplementary Fig 1). The disease was observed in 2 hectare model orchard with incidence of 45% causing leaf defoliation and thereby reducing the yield and quality of fruits. Transverse section cutting of algal spots revealed the algal thalli at subcuticular region and causing necrosis of epidermal cells. Sporangiophores (n=20) raised from algal leaf spot were cylindrical, 4 to 5 celled, 200-450 µm long x 8-20 µm wide, and forming a head cell with suffultory cells and sporangia on the top. Sporangia (n=20) were spherical to elliptical, rusty brown and 17.5-29 µm long × 18-23.6 µm wide and the total number of sporangia produced by each sporangiophores varies from 1 to 6. Setae (n=20) were filamentous with three to six celled, 17.5-50 µm long × 2.5-7.5 µm wide (Supplementary Figure 2). In our collection, mature gametangia were not observed. Morphological characters were studied on 20 diseased leaf samples collected from randomly selected five plants. To isolate pathogen, fresh algal thalli (n=5) were scraped from host tissue, surface sterilized (70% alcohol (30 s), 1% sodium hypochlorite (30 s) and sterile distilled water (3 × 60 s), inoculated to trebouxia liquid media and incubated at 25 ± 2 °C with a 12 hours photoperiod for 72 hours (Vasconcelos et al. 2018). The resultant five algal filaments were subjected to PCR amplification. The primer pair PNS1/NS41 was used in a PCR to amplify a fragment of 18S rRNA (Davis and Kaur 2019). The 18S rRNA gene sequences of the algae were compared using the Basic Local Alignment Search Tool (BLAST; http://www.ncbi.nlm.nih.gov/Blast/Blast.cgi) showed that our partial sequence had 99.5% similarity to C. virescens (KM020142.1). Hence, it was classified as C. virescens and sequences was deposited in NCBI-GenBank with accession numbers (OR053653, OR243777, OR429406, OR429407 and OR243779). For proving pathogenicity, algal filaments obtained from trebouxia liquid media were inoculated to 6 months old healthy A. carambola plant. Pathogenicity test was negative and typical symptoms could not be produced even up to 150 days of inoculation. In previous studies also, due to difficulty with production of zoospores in synthetic media, Koch's postulates of C. virescens as a plant pathogen has not been demonstrated experimentally (Sunpapao et al. 2017; Sanahuja et al. 2018; Kumar et al. 2019). In the second experiment, zoosporangia spore suspension were prepared from small pieces of algal leaf spot tissue processed in a sterile pestle and mortar and filtered through sterile cheesecloth (Sunpapao et al. 2017). A total of five isolates of zoosporangia spore suspension (1 x 102 to 1 x 104/ml of water) was sprayed on healthy, surface sterilized leaves of A. carambola plants (n=5) until runoff with a handheld airpump sprayer and incubated in green house (T: 25 oC, H: 80%). During the experiment leaves were remain attached to plant (5 days old) and plants were 6 months old grown in plastic pots under controlled conditions. Two plants were inoculated with each isolate and three non inoculated control plants were included. Non inoculated controls were sprayed with sterile distilled water. The pathogenicity experiment was repeated. The initial symptoms were produced 60 days after inoculation and complete algal thalli was observed on 90 days after inoculation, control plants were without any symptoms upto 150 days. Reisolated algal thalli from symptomatic plants were morphologically similar to original algal thalli and molecularly identified as C. virescens (accession number OR067193 and OR243810). Red rust caused by C. virescens is a major algal disease in the world and causing severe leaf defoliation in various horticultural crops viz., Mangifera indica (Vasconcelos et al. 2018), Manilkara zapota (Sunpapao et al. 2017), Psidium guajava (Rajbongshi et al. 2022), Ziziphus mauritiana (Shareefa et al. 2022) and Anacardium occidentale (Dooh et al. 2022). The available literature suggest that, this is the first report of algal leaf spot on A. carambola caused by C. virescens in India. This report extends the range of known pathogens associated with A. carambola plant and serves as a basis for development and implementing disease management strategies.

Effects of Truffle Inoculation on Root Physiology and Mycorrhizosphere Microbial Communities of Carya illinoinensis Seedlings

Although they are a valuable edible ectomycorrhizal fungus, truffles (Tuber spp.) nevertheless face significant difficulties in the development of their scale. As a type of high economic value nut-like economic forest tree, the pecan (Carya illinoinensis) serves as a natural host for truffles. However, the technology for mycorrhizal synthesis in pecan has not yet been developed, and it is still unclear how certain microbes affect this process. In this study, we inoculated the pecan root system with a suspension of truffle spores and investigated the growth physiology of pecan seedlings with various infestation levels, as well as changes in the soil’s physicochemical properties and the makeup of the microbial community at the root level. The findings showed that tuber inoculation significantly increased the peroxidase activity of the pecan root system, significantly decreased the pH, and effective phosphorus content of mycorrhizosphere soil, while increasing the nitrate nitrogen content, and significantly increased the abundance and diversity of the mycorrhizosphere soil fungal community. Different groups of fungal and bacterial markers were formed in the mycorrhizosphere of pecan seedlings at different levels of infestation. In the highly infested group, Rozellomycota and lasiosphaeriaceae were the difference marker fungi, and Xanthobacteraceae, Rhizobiaceae as well as Streptococcaceae were the difference marker bacteria. In the low-infestation group, sphaerosporella was differential marker fungi, and Bacillus and Tumebacillus were differential marker bacteria. The fungal marker flora of the control group consisted of Chaetomium and Gilmaniella. Pseudomonas was the marker bacterial community. Additionally, these fungi included Collarina and Rozellomycota, and several bacteria from the genera Pseudomonas, Gemmatimonas, and others showed highly significant relationships with changes in soil pH, effective phosphorus, and nitrate nitrogen. In conclusion, pecan–truffle mycorrhizal seedlings have the potential to create the ideal microbial community structure needed for mycorrhizal growth, and these microorganisms have the potential to significantly alter the pH, effective phosphorus content, and nitrate nitrogen concentration of the mycorrhizosphere soil. Our results contribute to the understanding of how the mycorrhizosphere microbial community evolves when exogenous mycorrhizal fungi infest host plants and can offer some theoretical guidelines for growing pecan–truffle mycorrhizal seedlings.

Alleviative effects of quercetin of Botrytis cinerea-induced toxicity in zebrafish (Danio rerio) larvae

Quercetin is a kind of flavonoid substance extensively existing in the plant, which has antioxidant, anti-inflammatory, and anti-apoptosis effects. It was reported that the higher concentration of spores present in the environment could cause abnormal development in zebrafish larvae. Therefore, this study set out to investigate whether quercetin could reduce the zebrafish larvae damage caused by Botrytis cinerea exposure as well as to examine the molecular basis for this action. The findings demonstrated that 50 μM quercetin improved the developmental dysplasia of zebrafish larvae induced by 102 CFU/mL Botrytis cinerea spore suspension, reduced abnormal apoptosis, enhanced antioxidant system, relieved inflammation, reshaped intestinal morphology and recovered intestinal motility. At the molecular level, quercetin decreased the transcriptional abundance of pro-apoptotic factors (bax, p53, caspase3, and caspase9) and up-regulated the anti-apoptotic gene (bcl-2) expression to reduce apoptosis. Moreover, quercetin enhanced the activities of downstream antioxidant enzymes (SOD and CAT) to clear excess ROS and MDA due to Botrytis cinerea exposure by up-regulating the expression of antioxidant genes (nrf2, ho-1, sod, and cat) in the Keap1-Nrf2 pathway. Additionally, quercetin inhibited the elevation of TNF-α by regulating the gene expression of key targets (jak3, pi3k, pdk1, akt, and ikk2) and the content of major proteins NF-κB (P65) and IκB in the NF-κB pathway. In conclusion, this work enriched the contents of the biological research of Botrytis cinerea and provided a new direction for the drug development and targeted therapy of quercetin.

Identification and biocontrol potential evaluation of a naturally occurring Metarhizium pingshaense isolate infecting tea weevil Myllocerinus aurolineatus Voss (Coleoptera: Curculionidae)

BackgroundTea weevil, Myllocerinus aurolineatus Voss (Coleoptera: Curculionidae), is an important insect pest in Chinese tea plantations. The primary method for controlling tea weevils involves the use chemical pesticides. Hence, there is an urgent need for environmentally friendly control strategies. To screen for potential pathogenic strains useful for the biocontrol of tea weevils, a naturally occurring Metarhizium pingshaense strain was isolated from a field-collected infected tea weevil larva for the first time in China.ResultsMorphological features and molecular characteristics revealed the isolate was an M. pingshaense strain, herein referred to as Ma0628. At 22 °C (tea weevil pupation temperature), the inoculation with M. pingshaense Ma0628 resulted in a corrected cumulative late instar larval mortality rate exceeding 76% at 11 days after the inoculation with the 1 × 108 conidia/ml spore suspension using the immersion or soil-mixing method. Accordingly, the median lethal concentrations were 4.49 × 103 and 3.76 × 102 conidia/ml for the immersion and soil-mixing inoculation methods, respectively. Furthermore, the corrected cumulative adult mortality rate reached 83.33% at 14 days after the inoculation with the 1 × 108 conidia/ml spore suspension.ConclusionThe study results indicate that M. pingshaense strain Ma0628 is an entomopathogenic fungus pathogenic to tea weevil larvae and adults, suggesting it may be a potentially useful biocontrol agent for preventing M. aurolineatus infestations.

CA 컨테이너를 이용한 수출 멜론의 선도유지 효과

This study investigates the effectiveness of CA (controlled atmosphere) containers in maintaining the freshness of exported melons. The melons were harvested on June 5, 2023, in the Yeongam area of Jeollanam-do, Korea. The CA container was loaded with melon samples packed in an export box. The temperature inside the container was set at 4°C, while the gas composition was set at 5% oxygen, 12% carbon dioxide, and 83% nintrogen. Following two weeks of simulated transportation, quality analysis was conducted at 10°C. The melons were inoculated with spore suspensions, and the decay rate was determined to investigate the effect of the gas composition inside the CA container on suppressing the occurrence of Penicillium oxalicum in melons. The results were compared with a Reefer container set at the same temperature. The samples transported in the CA container exhibited lower weight loss. The melon pulp softening, respiration rate, and ethylene production were slower using the CA container. Moreover, the decay rate during the distribution period in the CA container was lower than in the Reefer container. In contrast, the firmness of melons transported in the Reefer container decreased significantly (from 9.03N to 5.18N) immediately after transportation. The soluble solid content (SSC) of melons transported in the Reefer container also decreased rapidly. The results suggested that the CA container is the optimal export container for maintaining the freshness of melons.

First Report of Cladosporium ramotenellum Causing Sooty Spot on Postharvest Clementines in the United States.

In 2022, post-harvest symptoms of black spots were observed at an incidence of 2-5% on easy peeling clementines (citrus × clementina) in a fresh fruit market in Phoenix metropolitan area, Arizona. Black lesions on the fruit rind were superficial, circular, dry, and firm with gray sporulation. Gray or black aerial mycelium was also noticed on top of the black spots. Black spots were distributed over the entire fruit surface without any regular patterns. Maceration of rind and flesh was also noticed under refrigerated storage conditions. Two isolates were obtained (S13 and S14) and cultured on potato dextrose agar (PDA) at 25oC for 14 days. The colony color and texture of both isolates were identical on PDA: olivaceous black with abundant sporulation, margin entire edge to slightly undulate. Conidia were 0-1 septate and globose or ellipsoid with sizes ranging from 4-10 × 2-4 μm (n = 15). Ramoconida were also 0-1 septate and ellipsoid to cylindrical with sizes ranging from 15-30 × 2-5 μm (n = 15). Conidiophore had a width of 3-6 μm (n = 15). Isolates were identified as Cladosporium ramotenellum based on these morphological features (Bensch et al. 2012). The IDs of these two isolates were further confirmed by genomic DNA isolation, PCR amplification and sequencing of the ITS1-5S-ITS2-28S region of rDNA (V9G/LR5 de Hoog and Gerrits van den 1998, primers ITS4/ITS5 White et al.), actin gene (ACT-512F/ACT-783R, Carbone and Kohn 1999), and elongation factor 1α gene (EF1-1018F/EF1-1620R, Stielow 2015). Sequences of the two isolates were identical and thus only one sequence of each gene was deposited in the GenBank. A BLASTn search of actin sequence (233-bp, OQ185511) revealed 99.1% match with ex-type sequence EF 679538 (strain CBS:121628) of C. ramotenellum holotype (query coverage: 97%). BLASTn analysis of a portion of EF-1α gene (579-bp, OQ185512) revealed more than 99.7% similarity with sequences KU933429 (ATCC strain 16022) and MT881827 (strain 18EPLE003) of C. ramotenellum (query coverage: 100%). The ITS sequence (1519-bp, OQ236707) was identical to the ITS sequences of C. ramotenellum strains in easy peeler mandarins from Peru (Murciano et al. 2021). Pathogenicity tests were carried out twice on fresh easy-peeling clementine fruit. The inocula (1 x 105 spores/ml) were prepared in sterile distilled water containing 0.1% Tween 20 (TW) by mixing the conidial suspensions of two isolates from 7-day-old PDA cultures. Ten fruit were washed, surface sanitized with 70% ethanol, and wound-inoculated by immersing five fruit in spore suspension for 1 min. Five control fruit were wound-inoculated with TW. Inoculated and control fruit were stored in separate zip-lock bags for one week. Black spots resembling those observed on naturally infected fruit were present on inoculated fruit, while control fruit remained symptomless. C. ramotenellum was reisolated and was morphologically identical to the original isolates, thus completing Koch's postulates. C. ramotenellum has been reported as a fungal pathogen causing sooty spots on easy peeler mandarins from Peru (Murciano et al. 2021). To our knowledge, this is the first report of C. ramotenellum causing postharvest sooty spot and decay on clementines in the United States. Results show that infected fruit is potentially a pathogen source for long-distance dispersal. This occurrence was communicated to the state regulatory agencies for regulatory actions on imports of citrus fruit from countries with the occurrence of C. ramotenellum.

Beauveria bassiana (Balsamo) Vuillemin (Deuteromycota: Hyphomycetes) izolatlarının spor süspansiyonu ve kültür filtratının farklı koşullarda Tetranychus urticae Koch, 1836 (Acari: Tetranychidae) üzerindeki etkinliği

Abstract In this study, the effectiveness of Beauveria bassiana (Balsamo) Vuillemin (Deuteromycota: Hyphomycetes) isolates on Tetranychus urticae Koch, 1836 (Acari: Tetranychidae) was determined with Petri and pot experiments in laboratory and climate room conditions by applying spore suspension and culture filtrate in Isparta University of Applied Sciences of in 2022. In the Petri experiment, the spore suspensions (1x106, 1x107, and 1x108 spore/ml) and culture filtrates (1X, 5X, 10X) of BIM-001 and BY2 isolates of B. bassiana were sprayed in the form of mist for 10 seconds at a speed of 3 m/s onto leaf discs in Petri which included ten adult individuals. In the pot experiment, the same treatments were conducted with the same application method in the Petri experiment on single-leaf plants in pots. In the Petri and pot experiments, the difference between the mortality rates in the 108 spore/ml dose of BIM-001 and BY2 (64.00±4.52 and 42.00±2.49%, 58.00±2.91 and 41.00±2.77%, respectively) was significant on the 7th observation day. In the pure culture filtrate treatments, the mortality rates in the BIM-001 and BY2 were 73.00±2.13 - 68.00±3.59% and 60.00±3.65 -57.00±5.17% respectively in the Petri and pot experiments. BIM-001 and BY2 culture filtrate in both Petri and pot experiments (1X) applications were not statistically significant. The BIM-001 isolate of B. bassiana was more effective than the BY2 isolate treatment against adult females of T. urticae both in vivo and in vitro. In conclusion, it is thought that the culture filtrate of B. bassiana BIM-001 isolate is considered to have potential for the control of two-spotted spider mites.

Isolation, Behavioral Identification, and Pathogenicity Assessment of Entomopathogenic Fungi from a Forest Wood Borer.

Forest wood borers (FWB) cause severe tree damage and economic losses worldwide. The release of entomopathogenic fungi (EPF) during the FWB emergence period is considered an acceptable alternative to chemical control. However, EPF resources have been significantly less explored for FWBs, in contrast to agricultural insect pests. This paper presents a protocol for exploring EPF resources from FWBs using wild Monochamus alternatus populations as an example. In this protocol, the assignment of traps baited with M. alternatus attractants to different populations guaranteed the collection of adequate samples with natural infection symptoms, during the emergence periods of the beetle. Following finely dissecting integuments and placing them onto a selective medium, fungal species were isolated from each part of beetle bodies and identified based on both molecular and morphological traits. Several fungal species were certified as parasitic EPFs via re-infection of healthy M. alternatus with spore suspensions. Their behavioral phenotypes on M. alternatus were observed using scanning electron microscopy and further compared with those on the Coleopteran model insect Tribolium castaneum. For EPFs that present consistent parasitism phenotypes on both beetle species, evaluation of their activities on T. castaneum provided valuable information on lethality for future study on M. alternatus. This protocol helped the discovery of EPF newly reported on M. alternatus populations in China, which could be applied as an efficient approach to explore more EPF resources from other FWBs.

Dalbergia odorifera, a new host of Colletotrichum tropicale causing anthracnose in China.

Dalbergia odorifera T. Chen (Family: Fabaceae) is an endangered, wild and second class key protected tree species in China. It has been cultivated widely in south China because of its valuable rosewood and pharmacological uses. During January and September 2022, anthracnose was observed on D. odorifera in Chikan, Zhanjiang, Guangdong (N21°15' 9.4" E110°22' 25.9"). All of the D. odorifera plants were infected and the leaf disease incidence was above 30%. The lesions were 0.35 to 0.65 cm in size, greyish-white, and gradually expanded into brown black circular spots surrounded by a yellowish halo. Later, the centers of spots were white with dark-brown borders. In severe cases, the leaves turned yellow, withered and fell off. To isolate the pathogen, about 5×5 mm tissues at the disease-health junction of spots were sterilized with 75% ethanol for 30s and 3% hydrogen peroxide for 3 min, rinsed with sterile water three times, and plated onto PDA for incubation at 25-28℃ in the dark. Isolates were obtained by picking the tip of hypha growing from the tissues, further purified by coating the diluted spore suspension to obtain single-spore isolates. Colonies with abundant aerial mycelium were white to light gray, 4.0 to 5.0 cm in diameter after 5 days at 25 to 28℃ in the dark. Conidiogenous cells were hyaline, cylindrical and monoblastic. Conidia were unicellular, subcylindrical, hyaline, guttulate, 3.5 to 5.6×11.1 to 16.8 (av. 4.6±0.48 × 14.0±1.25) μm (n>50). Appressoria were dark brown subglobose, clavate, fusiform, occasionally lobed, terminal, 7.8 to 16.3×4.6 to 8.9 μm. No swollen cells and setae were observed. To further confirm identity of the fungus, partial regions of six genetic loci of isolate ACCC 35246 (stored in Agricultural Culture Collection of China) were amplified and sequenced: actin (ACT), chitin synthase (CHS), glyceraldehyde-3-phosphate dehydrogenase (GPDH), histone (HIS), beta-tubulin (TUB) and internal transcribed spacer (ITS) utilizing the primer pairs (Damm et al. 2012; Weir et al. 2012; White et al 1990) (OP314900-OP314904 and OP269660, respectively). Based on a BLAST analysis, the six sequences were about 99% identical to those of Colletotrichum tropicale holotype strain CBS124949 (ACT: JX009489 270/272; CHS: JX009870 295/299; GPDH: JX010007 274/279; HIS: KY856395 638/670; ITS: JX010264 546/548 and TUB: JX010407 494/497, respectively) (Weir et al. 2012). Phylogenetic analyses, using a combined dataset of ACT, CHS, GPDH, ITS and TUB were carried out in MEGA X using the maximum likelihood method, placed the fungus within the C. tropicale clade. Based on morphogical and molecular analyses, the fungus was identified as C. tropicale (Rojas et al 2010). To test the pathogenicity, five healthy leaves of 1 year-old potted plants were stab-wounded with a sterile needle and inoculated with 10 µL of spore suspension (105 conidia/mL). Another five healthy leaves were inoculated with sterile water as the control. Symptoms expressed by the inoculated leaves, 10 days after inoculation, were the same as those of the diseased plants, while the non-inoculated leaves remained asymptomatic. The same fungus based on morphogical and molecular criteria was re-isolated from the spots. As an endophyte or plant pathogen, C. tropicale inhabits a wide host range of plant species belonging to 31 genera in 25 families (Farr and Rossman 2022), D. odorifera is a new host of C. tropicale causing anthracnose in China. This work will be helpful for the diagnosis and control of this disease.

First Report of Panax notoginseng Wilt Disease Caused by Fusarium graminearum in China.

Panax notoginseng is one of the important economic crops under the forest, which is widely planted in Yunnan Province, China. In August of 2022, a survey in Xundian county (25º26' N, 12 103°7' E), was accomplished to verify the occurrence of wilt disease in P. notoginseng and understand its aetiology. The site is an underforest of organic P. notoginseng, covering an area of over 40 ha. Disease symptoms included severe stunting, leaf chlorosis, red or yellow stalks, and rotting roots. The entire plant gradually wilted and died with disease progression (Fig. 1). To identify the causal agent, we collected more than 30 wilted P. notoginseng plants and got the plant tissues from the symptomatic leaves, stalks, and roots. The tissues surface sterilised with 0.5% sodium hypochlorite for 2 min, followed by 75% alcohol for 1 min, and rinsed in sterilised water three times. Upon drying, samples were placed onto potato dextrose agar (PDA) incubated in the dark at 25°C (Bilgi et al. 2011). Isolates were then transferred to carnation leaf agar (CLA) to induce sporulation. Colonies on PDA were yellow, orange to red, with abundant fluffy aerial mycelia with a dark red pigment on the undersides; Colonies on CLA were orange to yellow (Fig. 2). Fusiform macroconidia and bottle-shaped conidiogenous cells were visible under a microscope. Microconidia were not observed. Macroconidia were measured as 18.5-40.5 μm × 3-4.7 μm (n = 60) (Fig. 3), and possessed 2 to 6 septa. These are similar to previously reported morphological characteristics of Fusarium graminearum (Shikur et al. 2018; Martinez et al. 2019). Cetyltrimethylammonium bromide rapid plant genome extraction kit-DN14 was used to obtain genomic DNA from two representative isolate, the ITS, TEF1 and RPB2 gene were amplified by Polymerase Chain Reaction using primers ITS5/ITS4 (White et al, 1990), EF1-983F/EF1-2218R (Rehner et al, 2005), bRPB2-6F/bRPB2-7.1R (Matheny et al, 2002), respectively. BLAST homology search for nucleotide sequences revealed > 99% similarity to F. graminearum ITS (550bp; MG274308, KU847854), TEF1 (1000bp; MH572248, MH572252) and RPB2 (1000bp; KT855203, KT855206) sequences. All sequences from this study were deposited in GenBank (OP617343 and OP617344 for ITS; OP930951 and OP930952 for TEF1; OP930953 and OP930954 for RPB2). In the phylogenetic tree, the isolates (SWFU 0000116, SWFU 0000117) clustered with the representative strains of F. graminearum. The morphology and multi-gene phylogenetic analysis indicated that the new isolate is F. graminearum. Koch's postulates were used to confirm that the symptoms in wilted P. notoginseng were attributable to F. graminearum. First, healthy leaves were gently wounded with a needle and sprayed with spore suspension (1.0 × 106 spores mL-1) in a hand sprayer (Martinez et al, 2019). All P. notoginseng plants were then replanted in pots with a diameter of 20 cm (1 plants/pot) filled with mixture of sterilised soil, and incubated at 25-27°C. The blank control comprised sterile cotton soaked in sterile water and inactivated mycelia sprayed on the leaves. After 7d of incubation, all inoculated leaves and stalks developed necrosis and developed pale red mycelia, while control plants remained symptomless (Fig. 4-5). The pathogen was successfully isolated from these inoculated plants and identified as F. graminearum. Koch's postulates were implemented. To the best of our knowledge, this is the first report from China with evidence of F. graminearum infecting P. notoginseng.

First Report of Pseudocercospora paraguayensis Causing Leaf Spots on Bixa orellana from Chinese Mainland.

Bixa orellana L. is a traditional Chinese medicine. In December 2019, a leaf spot disease was observed on B. orellana from a field in Zhanjiang (21°18'12''N, 110°17'22''E), China. Disease incidence was around 85% (n = 100 investigated plants from about 30 hectares).Initial leaf spots were circular, and the center of the lesions was grayish-white with a purple black border. The coalescence of individual spots eventually led to leaf wilt. Ten symptomatic leaves from 10 plants were sampled. The margins of the samples were cut into 2 mm × 2 mm pieces, and the surfaces were disinfected with 75% ethanol for 30 sec, and 2% sodium hypochlorite for 60 sec. The the samples were then rinsed three times in sterile water, plated on potato dextrose agar (PDA), and incubated at 28 °C. Pure cultures were obtained by transferring the hyphal tips to new PDA plates. Three representative isolates (BOPP-1, BOPP-2, and BOPP-3) were used for further study. The colonies of isolates on PDA were dark olive green with off-white aerial mycelia after 7 days at 28 °C. Conidia were solitary, smooth to verrucous, olive to light brown, slightly curved, narrowly obclavate, apex obtuse, base obconic-truncate, had 2-4 septa, and 30.4-55.5×2.0-3.5 μm in size.. These morphological characteristics showed did not differ from the description of Pseudocercospora paraguayensis (Crous et al. 1997). For molecular identification, the internal transcribed spacer (ITS) region, the translation elongation factor 1-α (TEF1) gene, and actin (ACT) gene were amplified using primer pairs ITS1/ITS4 (White et al., 1990), EF1/EF2 ( O'Donnell et al. 1998), and ACT-512F/ACT-783R (Carbone and Kohn, 1999) and sequenced from DNA extracted from the three isolates, respectively. Sequences were deposited in GenBank under accession no. MZ363823-MZ363825 (ITS), MZ614954-MZ614956 (TEF1), and MZ614951-MZ614953 (ACT). A phylogenetic tree was generated on the basis of the concatenated data from the sequences of ITS, TEF1, and ACT that the three isolates were nested within the clade containing the type specimen of P. paraguayensis (CBS 111286) but not within P. bixae (the type specimen CPC 25244). Pathogenicity was tested through in vivo experiments. Inoculation and control seedlings (n = 5, 1-month-old) were sprayed with a spore suspension (1 × 105 per ml) of P. paraguayensis and sterile distilled water (control), respectively, until run-off (Fang. 1998). The plants were grown in pots in a greenhouse at 28°C, with at approximately80% RH. The test was performed three times. Symptoms similar to those in the field were observed on the inoculated plants after two weeks. The control plants remained healthy. The fungus was re-isolated from the infected leaves and confirmed as the same isolatesby morphological and comparison of ITS sequences with 100% identical to those of isolates. No original fungi were isolated from the control plants. A previous study reported that P. paraguayensis caused leaf spots on pistachio and eucalypts, and the fungus causing the leaf spots of B. orellana was redescribed as P. bixae (Crous et al. 2019). However, multilocus phylogenetic analyses differentiated P. paraguayensis from P. bixae. In the present study, P. paraguayensis was distinguished from P. bixae due to the absence of catenulate conidia and the presence of finely verruculose conidia (Crous et al. 2013). P. eucalypti as a synonyms was reported in Taiwan (www.MycoBank.org). The current study is the first to report P. paraguayensis causing leaf spots on B. orellana from Chinese Mainland. This finding will help to provide a scientific basis for the disease detection.

In-vitro Analysis of Different Trichoderma spp. Against Fusarium Rhizome Rot of Ginger (Zingiber officinale Roscoe)

This study evaluated the effectiveness of four isolates of Trichoderma spp. obtained from various plant protection divisions and commercial products for the biocontrol of Fusarium rhizome rot of ginger (Zingiber officinale Roscoe). Experiments were designed in a completely randomized design (CRD) with five treatments and ten replications. The National Agriculture Research Council (NARC) isolates of Trichoderma spp. were multiplied through direct inoculation on PDA media. Meanwhile, the commercial products of T. harzianum (TRICHO-HR) and T. viride (Biocide Trivi) were prepared as spore suspension. The dual culture technique was used to evaluate the interaction between Trichoderma spp. and Fusarium pathogen. The radial diameter of both Trichoderma and Fusarium was measured every 24 h for 7 days. The experiments in dual cultures demonstrated that various Trichoderma isolates and commercial products significantly inhibited Fusarium mycelial growth. Among the isolates tested, T1-NARC showed the highest efficacy at 57.91% growth inhibition, followed by T2-RPPL (52.50%) and T4-Biocide Trivi (40.98%). However, T3-TRICHO HR displayed lower performance with an inhibition rate of 24.75%. These findings offer valuable insights for the use of Trichoderma spp. as biocontrol agents in ginger cultivation, contributing to improved disease control and enhanced crop health. The observed differences in performance could be attributed to the genetic variations among the isolates.

Endophytic colonization of sugar beet by Beauveria varroae and Beauveria bassiana reduces performance and host preference in army worm, Spodoptera littoralis

Spodoptera littoralis (Lepidoptera: Noctuidae) Boisduval 1833 is a voracious leaf feeder and major agricultural pest attacking numerous crops. Development of insecticide resistance hampers management efforts for S. littoralis and entomopathogenic fungi represent one possible alternative management tool. Here, we quantified colonization of sugar beet, Beta vulgaris L, with two fungal entomopathogens, Beauveria varroae Vuillemin 1912 and Beauveria bassiana (Balsamo) Vuillemin 1912 (Ascomycota: Hypocreales), and examined their endophytic effects on biological and reproductive parameters of S. littoralis. Two methods were used to inoculate sugar beet: soaking seeds in spore suspension and spray application of conidia onto leaves. For both methods, fungal suspensions with concentrations of 1 × 104 and 1 × 108 conidia/ml colonized roots, stems, and leaves of sugar beet. The colonization rate increased over time. Following inoculation of seeds, weight gain of S. littoralis larvae fed on leaves infected with endophytic fungi was reduced compared with controls. Furthermore, protease and lipase activities in the gut of S. littoralis decreased with increasing concentration of fungus applied to plants. An increase in phenol oxidase activity in the gut also indicated increased defense response of larvae feeding on Beauveria-infected leaves. Both B. bassiana and B. varroae decreased the duration of larval and pupal stages, adult emergence, and oviposition by females. Incubation of sugar beet seeds with B. varroae or B. bassiana decreased survival of pupae and egg laying by female moths on endophyte-infested treatments by 30 and 50%, respectively, compared with controls. Furthermore, an initially female biased sex ratio decreased toward 50:50 over time in populations of S. littoralis reared on sugar beet colonized by fungal endophytes as compared with controls, which remained female-biased. Endophyte inoculation increased growth of sugar beet compared with controls. Our results indicate that B. bassiana and B. varroae affect survival of S. littoralis and may be useful for management of this pest.

First report of Fusarium clavum causing garlic bulb rot in Mexico.

In Mexico, the main states for garlic (Allium sativum L.) production are Zacatecas, Guanajuato, and Puebla. In the 2020 crop season, garlic cultivation encompassed 6,794 ha, yielding 85,505 tons (SIAP, 2021). In February 2020, 35 garlic samples showing basal rot symptoms were collected from the garlic-growing regions in the states of Zacatecas and Aguascalientes in the municipalities of San Antonio Tepezala 22°13'13.5''N, 102°15'55.3''W, Rincón de Romos 22°17'44.9''N, 102°13'06.8''W, and Calera 22°58'39.4''N and 102°41'29.9W, respectively. The sampling carried out was random sampling by conglomerates, dividing each field in groups with plants that showed similar symptoms. The infected plants were stunted in growth, with reddish dying leaves. The stalks and bulbs were soft, and their root system was poorly developed. The collected samples were placed in polyethylene bags and taken to laboratory. The roots and bulbs of 35 plants were cleaned, portions of the diseased tissue was cut into 0.5 cm pieces and disinfected in 1% sodium hypochlorite for 3 minutes. The samples were rinsed twice with sterile distilled water and dried on sterile paper towels. The tissues were cultured on Potato Dextrose Agar (PDA) medium and incubated in the dark at 25°C. Seven days after incubation, pure cultures were obtained using monoconidial cultures technique on Spezieller Nährstoffmmarmer agar (SNA) and subcultured on carnation leaf agar (CLA). Ten isolates were obtained that grew slowly, showing a white coloration, then turning yellow with abundant aerial mycelia. Microscopic traits of 30 characterized spores included slender macroconidia that were curved dorsiventrally, tapering towards both ends, with five to seven thin septa, measuring 36.4-56.6 µm × 4.0-4.9 µm in size and chlamydospores that were abundant, globose to oval, subhyaline and terminal or intercalary in chains measuring 8.8-4.5 µm in diameter. Microconidia, were single-celled, hyaline, nonseptate, and ovoid. The morphological traits matched the description of Fusarium clavum (Xia et al. 2019). To confirm the strain's identity, DNA was extracted from six monoconidial cultures and used as template to amplify translation elongation factor (TEF) gene 1α, RNA polymerase largest subunit (RPB1), and RNA polymerase second largest subunit (RPB2) (O'Donnell et al. 2010). The products were sequenced and deposited in GenBank as ON209360, OM640008 and OM640009, the homology analysis using BLASTn was similar to F. clavum with 99.46%, 99.49% and 98.82% respectively with E VALUE 0.0 in all cases with access numbers OP48709, HM347171 and OP486686. Koch postulate was performed to confirm the pathogenicity of the six isolates. Variegated garlic cloves were planted after being disinfected with sodium hypochlorite at 3% w/v in 2-kg pots under the greenhouse conditions. When the garlic plants developed 4 or 5 true leaves, their basal stalks were inoculated by pouring uniformly with 1 mL of a spore suspension at 108 conidia/mL prepared from 1-week-old colonies (Lai et al. 2020). Twenty-four plants were inoculated with six isolates (four plants per isolate), and four control plants were treated with sterile distilled water. Symptoms appeared 20 days post-inoculation. The leaves were reddish, and the stalks were soft. The leaves eventually developed foliar dieback disease symptoms, their root system showed brown lesions and rot, and all water-inoculated controls remained asymptomatic. Isolations were made on the diseased plants, and the inoculated pathogen was recovered and confirmed morphologically and molecularly by DNA extraction and PCR reactions. Koch's postulate was repeated twice, obtaining the same results. To our best knowledge, this is the first report of F. clavum infecting Allium sativum L. in Mexico. bulb rot caused by F. clavum is a severe threat to garlic cultivation, and identification of this pathogen is important for effective disease management and control.

Detached Maize Sheaths for Live-Cell Imaging of Infection by Fungal Foliar Maize Pathogens.

We have optimized a protocol to inoculate maize leaf sheaths with hemibiotrophic and necrotrophic foliar pathogenic fungi. The method is modified from one originally applied to rice leaf sheaths and allows direct microscopic observation of fungal growth and development in living plant cells. Leaf sheaths collected from maize seedlings with two fully emerged leaf collars are inoculated with 20 µL drops of 5 x 105 spores/mL fungal spore suspensions and incubated in humidity chambers at 23 °C under continuous fluorescent light. After 24-72 h, excess tissue is removed with a razor blade to leave a single layer of epidermal cells, an optically clear sample that can be imaged directly without the necessity for chemical fixation or clearing. Plant and fungal cells remain alive for the duration of the experiment and interactions can be visualized in real-time. Sheaths can be stained or subjected to plasmolysis to study the developmental cytology and viability of host and pathogen cells during infection and colonization. Fungal strains transformed to express fluorescent proteins can be inoculated or co-inoculated on the sheaths for increased resolution and to facilitate the evaluation of competitive or synergistic interactions. Fungal strains expressing fluorescent fusion proteins can be used to track and quantify the production and targeting of these individual proteins in planta. Inoculated sheath tissues can be extracted to characterize nucleic acids, proteins, or metabolites. The use of these sheath assays has greatly advanced the detailed studies of the mechanisms of fungal pathogenicity in maize and also of fungal protein effectors and secondary metabolites contributing to pathogenicity.

Biological control potential of a soil fungus Trichoderma asperellum K1-02 against Neoscytalidium dimidiatum causing stem canker of dragon fruit

Stem canker on dragon fruit caused by the fungus Neoscytalidium dimidiatum negatively impact dragon fruit cultivation and production. The use of Trichoderma species, the biological control agent against stem canker disease is an alternative way instead of use of synthetic fungicides. This study aimed to test the antifungal activities of soil fungi Trichoderma against N. dimidiatum. Ten isolates of Trichoderma species were tested against N. dimidiatum via dual culture assay, and the results showed that percentages of inhibition ranged from 72.75 to 84.45%. Trichoderma species K1-02 was found to be the most effective strain against N. dimidiatum (84.45%). Trichoderma K1-02 was identified based on a morphology and molecular study of internal transcribed spacer (ITS), the second largest subunit of RNA polymerase II (rbp2), and the translation elongation factor 1-alpha (tef1-α) as Trichoderma asperellum K1-02. The pre-colonized plate method demonstrated that T. asperellum K1-02 covered the tested plates at a rate of 60%, which was significantly higher than N. dimidiatum (40%), and T. asperellum K1-02 aggressively covered the mycelia of N. dimidiatum as observed via stereomicroscopy. An enzyme assay was conducted to test the activities of cell wall-degrading enzymes (CWDEs) in cell-free culture filtrate (CF) released by T. asperellum K1-02, and the results showed that the activities of chitinase and β-1,3-glucanase were significantly higher than those of the control (potato dextrose broth alone). Scanning electron microscopy of N. dimidiatum mycelia incubated with cell free CF of T. asperellum K1-02 showed abnormal morphology. Furthermore, an in vivo test was conducted on dragon fruit cladodes to test the ability of Trichoderma with respect to the reduction of canker lesions. The results revealed that the application of 106 spore/mL T. asperellum K1-02 spore suspension suppressed disease development via the reduction of canker areas (0.09 cm2) on dragon fruit cladodes at a significantly higher level than those inoculated by pathogens alone (0.49 cm2). Thus, T. asperellum K1-02 demonstrated antifungal abilities against N. dimidiatum and has the potential to control stem canker on dragon fruit.

Preparation and activity of sodium carboxymethyl cellulose (CMC-Na) and Metarhizium rileyi ZHKUMR1 composite membrane

Improving the adhesion capability of Metarhizium rileyi ZHKUMR1 on leaves enabled by the combination with Carboxymethyl Cellulose (CMCNa) materials is feasible to improve the utilization rate of Metarhizium rileyi. Herein, the CMC-Na-ZHKUMR1 membrane was prepared by simply mixing Carboxymethyl Cellulose (CMCNa) with Metarhizium rileyi. Through compatibility test, it was found that the inhibition rates of spore germination and mycelial growth of ZHKUMR1 were only 1.51 % and 3.13 % when the concentration of Carboxymethyl Cellulose (CMCNa) was 0.5 %. By adding 2 % of Carboxymethyl Cellulose (CMCNa) under UV irradiation for 30 min, the protective rate of spore germination of ZHKUMR1 was up to ~12.44 %, where the wettability on corn leaves was achieved and the retention of ZHKUMR1 spores on corn leaves was increased. After indoor activity determination, it was found that after 3 min of simulated rain washing, the lethal rate of corn leaves pretreated with CMC-Na-ZHKUMR1 on the 2nd instar larvae of Spodoptera frugiperda was 46.67 %, which was much higher than that of ZHKUMR1 spore suspension alone. This work clearly showed that Carboxymethyl Cellulose (CMCNa) effectively improved the field application effect of Metarhizium rileyi ZHKUMR1, and this strategy provided guidance for improving the field efficacy of Metarhizium rileyi ZHKUMR1.

<i>Fusarium equiseti</i> as one of the main <i>Fusarium</i><i></i> species causing wilt and root rot of chickpeas in Morocco

Fungal isolates of <i>Fusarium</i> were collected from symptomatic chickpea (<i>Cicer arietinum</i> L.) plants growing in fields within the Souk Tlat commune in the Gharb region. Morphological and molecular characterizations were performed of the fungal isolate N3 obtained from a chickpea plant. PCR amplification and sequencing of the internal transcribed spacer using the primers ITS1 and ITS4 were applied to identify the fungal isolate N3. The maximum similarity index of the fungus was found to be 99.33% with <i>Fusarium equiseti</i> (accession no. MT111122). In the pathogenicity test, both chickpea seed dip inoculation and soil infestation by the spore suspension of <i>Fusarium</i> isolate were adopted. Four weeks after chickpea seed inoculation, few plants emerged, and those that emerged were stunted. A high percentage of inoculated seeds did not emerge and showed accentuated rot symptoms. Eight weeks after sowing seeds in infested soil, the obtained chickpea seedlings displayed root necrosis, browning at the crown, and wilting. In addition, these plants showed a foliar alteration index of 0.395. The re-isolation was positive for different parts of chickpea plants for both seed and soil inoculation. <i>Fusarium equiseti</i> isolate decreased the length of the root and aerial parts, and the number of leaves and branches of the inoculated chickpea plants either by seed inoculation or soil infestation with values of 0.91 cm and 19.73 cm, 1.29 cm, and 19.44 cm, 1.11 and 18.66, and 0.0 and 2.08 respectively, whereas the corresponding values for the control plants were 27.16 and 28.33 cm, 29.05 and 31.05 cm, 24.21 and 25.66, and 3.50 and 3.11, respectively. To the best of our knowledge, this is the first report of <i>F. equiseti</i> on chickpea (<i>Cicer arietinum</i> L.) in Morocco.

Development of a novel pilot-scale tray bioreactor for solid-state fermentation aiming at process intensification

Solid-State Fermentation (SSF) processes have high contamination and low productivity rates, since it involves batch processes with multiple manual stages. In order to reduce handling in SSF processes, this research developed a pilot-scale tray bioreactor that enables process intensification by combining upstream operations and fermentation in a single equipment. Thus, a novel tray bioreactor was developed with an atomization system of spore suspension, nutrient solution, and cooling water above the fermentative medium. It also incorporates a sterilization system using steam. In order to validate the bioreactor, two case studies were conducted. In the first case study, the fungus Metarhizium anisopliae was cultivated on three types of agro-industrial residues. The conidia/mL obtained using the mixture of wheat bran and broken rice was (16.7±0.46)×108, which is statistically higher (α=0.05) than the fungus production of (8.59±1.60)×108 cultivated on the control (Type I Rice). In the second case study, lipases from M. anisopliae were produced on agro-industrial residues. The enzymatic activity average was 5.64±0.45U/mL based on 36 fermentations. The results have shown that the new tray bioreactor can produce the fungus and lipases in greater quantities than other systems reported in literature, while reducing handling, energy, costs, and process time.