Identification of Monilia species in Tibet and characterization of M. yunnanensis in China

Samples of peach and plum fruits with brown rot symptoms were collected from Tibet in 2019 and 2020, and the causal agent was identified as Monilia yunnanensis, which represents the first characterization of Monilia spp. on peach and plum in Tibet. Morphological investigation showed that some conidia from naturally diseased fruits were larger than those observed in previously isolated M. yunnanensis. Some conidia of M. yunnanensis isolates from Tibet produced more than two, even up to six germ tubes from different parts of each conidium, instead of one or two germ tubes developing from the pointy sides of each conidium. The alignment of ribosomal internal transcribed spacer region (ITS) sequences revealed that some isolates from Tibet displayed a mutation at the 374th position from adenine (A) to cytosine (C). Although above-mentioned differences were observed between isolates from Tibet and other regions, phylogenetic analysis indicated that all of the M. yunnanensis isolates from different stone fruits and different regions in China were clustered together without obvious genetic differentiation. These results revealed that hosts and geographical environments did not play a major role in the evolution of M. yunnanensis.

The infection process of Colletotrichum fuscum on oregano leaves and stems

Anthracnose, caused by Colletotrichum fuscum, produces regular necrotic spots on oregano leaves and stems, causing severe crop losses. In this study, Koch’s postulates were fulfilled and infection process was investigated using scanning electron microscopy. Leaves and stems of Origanum vulgare were inoculated and incubated at 24°C in wet chambers under high relative humidity. Pathogenicity experiments demonstrated that all tested C. fuscum isolates had infected stems and leaves of oregano. Of all inoculation methods, direct placement of colonized agar plugs on injured epidermis and soaking plant organs in conidial suspension were the most effective. The behavior of the conidia deposited on the oregano leaves was investigated at different time intervals after inoculation: at 12, 18, 32, 48, 67 and 98 h. Conidia produced an appressoria of varying shapes which has been formed at the end of germ tubes of different lengths. Penetration to host tissue through stomata was observed. Acervuli formed on the leaves surface after 98 h after inoculation, typically with sharp pointed setoses.

Cytological observation of the infection process of Venturia carpophila on peach leaves

Peach scab caused by Venturia carpophila, is one of the most destructive fungal diseases of peach worldwide, which seriously affects the peach production. Up to date, the infection process and pathogenesis of V. carpophila on peach remain unclear. Here, we present the infection behaviour of V. carpophila at the ultrastructural and cytological levels in peach leaves with combined microscopic investigations (e.g., light microscopy, confocal laser scanning microscopy, scanning electron microscopy and transmission electron microscopy). V. carpophila germinated at the tip of conidia and produced short germ tubes on peach leaf surfaces at 2 days post-inoculation (dpi). At 3 dpi, swollen tips of germ tubes differentiated into appressoria. At 5 dpi, penetration pegs produced by appressoria broke through the cuticle layer, and then differentiated into thick sub-cuticular hyphae in the pectin layer of the epidermal cell walls. At 10 dpi, the sub-cuticular hyphae extensively colonized in the pectin layer. The primary hyphae ramified into secondary hyphae and proliferated along with the incubation. At 15 dpi, the sub-cuticular hyphae divided laterally to form stromata between the cuticle layer and the cellulose layer of the epidermal cells. At 30 dpi, conidiophores developed from the sub-cuticular stromata. Finally, abundant conidiophores and new conidia appeared on leaf surfaces at 40 dpi. These results provide useful information for further understanding the V. carpophila pathogenesis.

Costunolide Influences Germ Tube Orientation in Sunflower Broomrape – A First Step Toward Understanding Chemotropism

Orobanche cumana WALLR. is a host-specific root parasite of cultivated sunflowers with increasing economic importance in Europe, North Africa, and parts of Asia. While sesquiterpene lactones (STLs) released from sunflower roots were identified as natural germination stimulants of O. cumana seeds in the soil, the chemical nature of the signals guiding the emerging germ tube toward the host root has remained unknown hitherto. Thus, we designed a bioassay that allowed the observation of broomrape germination and subsequent germ tube development in the presence of substances with putative chemotropic activity. Root exudates and sunflower oil extracts, both containing STLs in micromolar concentrations, caused the positive chemotropic orientation of germ tubes. A similar positive chemotropic effect was achieved with costunolide, one of the four STLs of sunflower present in the exudate and oil extracts. In contrast, GR24, a synthetic strigolactone (SL) with germination-inducing activity on O. cumana seeds, showed no effect on the germ tube orientation. The effect of costunolide was concentration-dependent and within the range of its natural micromolar occurrence in roots. We assume that an STL gradient is responsible for the stronger inhibition of elongation growth on the host-facing flank of the germ tube compared with the far side flank. This would confer a double role of STLs from sunflower root exudates in the sunflower–broomrape interaction, namely, as germination stimulants and as chemotropic signals.

First Report of Podosphaera aphanis Causing Powdery Mildew on Wild Blackberry Species (Rubus spp.) in Mexico

Wild blackberry species (Rubus spp. L.; Rosaceae) represents an invaluable source of genes for the generation of new varieties, but also serve as a primary source of disease inoculum. During April of 2020, symptoms of powdery mildew were observed on four populations of wild blackberry species located in the states of Chiapas (16°59'11"N, 92°59'07"W; 16°47'08"N, 92°31'05"W) and Michoacán (19°37'17"N, 100°08'59"W; 19°29'25"N, 101°32'54"W), Mexico. Signs of the pathogen were white powdery masses mainly on the top of new shoots. Symptoms included yellowing, necrosis, and early defoliation of the plants. Hyphae were tin-walled, hyaline, smooth, and 4.0–9.0 mm wide. Appressoria were indistinct -to- nipple-shaped. Conidiophores (n=30, 75–225 × 10.5–13.5 μm) were straight, and unbranched with cylindrical foot cells (n=30, 31.5–158 × 8–13.5 μm), straight, somewhat widening upwards, followed by 1–3 shorter cells. Conidia (n=100; 25.5–38.5 × 9.5–22.5 μm) were catenulate, ellipsoid-ovoid -to- doliiform, containing fibrosin bodies (in 3% KOH). Germ tubes (n=30, 13.5–40.5 × 4.5 μm) emerged laterally, and were unbranched with slightly swollen tips. Chasmothecia were not found. Morphological characters of the fungus in all samples corresponded to the previous descriptions of Podosphaera aphanis by Braun and Cook (2012) and Stevanovi´c et al. (2020). Voucher specimens were deposited in the Department of Agricultural Parasitology Herbarium at the Chapingo Autonomous University under accessions UACH421, UACH423, UACH425, UACH426. To confirm the species identification, the internal transcribed spacer (ITS) of one sample was amplified using the primers ITS5 (White et al. 1990) and P3 (Kusaba and Tsuge, 1995) and sequenced. The sequence was deposited in GenBank (accession number MW988591). A phylogenetic analysis using Bayesian inference and maximum likelihood was performed (Hernández-Restrepo et al. 2018) and included other Podosphaera species (Takamatsu et al. 2010). The sequence from the isolate UACH426 clustered with the strain MUMH1871 of P. aphanis forming a definite clade and remained as a sister taxon of P. pannosa. Pathogenicity was verified through inoculation by gently dusting conidia from one powdery mildew patch onto leaves of five healthy blackberry plants of each specie. The same number of noninoculated plants served as controls. All plants were maintained in a greenhouse at 25–30°C with 75% relative humidity. All inoculated plants developed powdery mildew symptoms after 12 days, whereas no symptoms were observed on noninoculated plants. The fungus recovered from the inoculated plants was morphologically identical to that originally observed on diseased blackberry plants, demonstrating the pathogenicity of the fungus. Based on morphological data and phylogenetic analysis, the fungus was identified as P. aphanis. This fungus has been reported to cause powdery mildew on blackberry plants in Serbia (Stevanovi´c et al. 2020). This is the first report of P. aphanis causing powdery mildew on wild backberry species in Mexico according to Farr and Rossman (2021). The primary source of inoculum of powdery mildew for commercial plantings is wild blackberry plants from noncultivated areas and may warrant control of wild populations.

Conservative production of galactosaminogalactan in Metarhizium is responsible for appressorium mucilage production and topical infection of insect hosts

The exopolysaccharide galactosaminogalactan (GAG) has been well characterized in Aspergilli, especially the human pathogen Aspergillus fumigatus. It has been found that a five-gene cluster is responsible for GAG biosynthesis in Aspergilli to mediate fungal adherence, biofilm formation, immunosuppression or induction of host immune defences. Herein, we report the presence of the conserved GAG biosynthetic gene cluster in the insect pathogenic fungus Metarhizium robertsii to mediate either similar or unique biological functions. Deletion of the gene cluster disabled fungal ability to produce GAG on germ tubes, mycelia and appressoria. Relative to the wild type strain, null mutant was impaired in topical infection but not injection of insect hosts. We found that GAG production by Metarhizium is partially acetylated and could mediate fungal adherence to hydrophobic insect cuticles, biofilm formation, and penetration of insect cuticles. In particular, it was first confirmed that this exopolymer is responsible for the formation of appressorium mucilage, the essential extracellular matrix formed along with the infection structure differentiation to mediate cell attachment and expression of cuticle degrading enzymes. In contrast to its production during A. fumigatus invasive growth, GAG is not produced on the Metarhizium cells harvested from insect hemocoels; however, the polymer can glue germ tubes into aggregates to form mycelium pellets in liquid culture. The results of this study unravel the biosynthesis and unique function of GAG in a fungal system apart from the aspergilli species.

Thevetia peruviana (Pers.) K. Schum. Potential Antifungal Agent Against Mycosphaerella fijiensis Morelet, Fungi Responsible of Black Leaf Streak Disease (BLSD) of Plantain (Musa spp)

Alternatives to synthetic chemicals are undertaken against phytopathogens. The aim of this work is to evaluate the effect of seed extracts of Thevetia peruviana (Pers.) K. Schum. on Mycosphaerella fijiensis Morelet, fungus responsible for banana black leaf streak disease. Five extracts of T. peruviana, hexane extract (HE), ethyl acetate extract (EAE), acetone extract (AcE), methanol extract (ME) and aqueous extract (AqE), and a fungicide, Azoxystrobin were used. GC-MS of acetone extract was performed. Fifty (50) strains of M. fijiensis per sampling site were tested. Three concentrations of extracts 6.25 (C1), 12.5 (C2), and 25 (C3) μl/ml, a negative control (0 μl/ml) and 10 ppm of azoxystrobin were used for the tests. The MIC50 and MIC90 were determined. GC-MS showed chemical compounds with different molecular height such as acids, sugars, and esters. AcE and AqE significantly reduced M. fijiensis germ tube growth at C2 and C3 concentrations and with inhibition percentage respectively ranged of 60-90% and 40-80%. The growth levels of the germ tubes were above the strobilurin resistance threshold at Njombe and peasant plantation, ranging from 77.9% to 92.3%. AcE showed the same or superior efficacy as the fungicide used on conidial germination at all tested concentrations. The MIC50 totally reducing mycelial growth and conidial germination was 6.25 μl/ml. T. peruviana seeds extracts can be exploited in integrated pests management against M. fijiensis.

First Report of Brown Rot of fruit on Vitis amurensis Caused by Monilinia polystroma in China

Vitis amurensis Rupr. (Amur grape) is a wild grape genetic resource widely distributed in Heilongjiang, Jilin, Liaoning, and Inner Mongolia, among other places in China (Song et al. 2009) and the Russian Far East and Korean Peninsula. In September 2018, brown rot symptoms were observed at ripening stage on the fruits of a 5-year-old Amur grapevine germplasm resources nursery of the cultivar ‘Beibinghong’ and a few Russian resources in Zuojia Town, Jilin City, Jilin Province, China. The diseased fruit surface became brown with soft rot and produced buff to brownish-grey sporodochia with conidia. Around 180 plants of ‘Beibinghong’ were examined which had 8 % incidence. Forty five samples were collected from symptomatic fruits of 15 randomly sampled ‘Beibinghong’ grape clusters, cut into 5-mm2 pieces of diseased tissue, surface sterilized with 1% NaOCl for 2 min, rinsed three times with sterile water, dried on sterilized filter paper, and plated on potato dextrose agar (PDA). Thirteen monosporic isolates were obtained using the single-spore isolation method with incubation at 25°C and a 12-h light/12-h dark cycle. The average colony diameter was 46–49 mm after 4 days of culture on PDA. Colonies were white to grayish with even margins. Irregular black stromatal crusts were observed on the reverse side of dishes 10 days after inoculation. Conidial spores were produced when cultured on cherry agar at 25°C under near-ultravolet light. Spores were single-celled and hyaline, limoniform or ellipsoid, and were produced in branched monilioid chains, 12–22 × 8–13 µm (mean: 15.4 ± 1.03 × 9.01 ± 0.72 µm, n = 50). When conidia were cultured on water agar at 25°C for 18 h, the germ tubes were straight, 700–1,000 µm long, and often with two germ tubes per conidium. Morphological characteristics were consistent with those of Monilinia polystroma (van Leeuwen et al. 2002). To confirm the species identification, two DNA regions of the selected isolate ‘VAMPWYZSH8’ were amplified by polymerase chain reaction (PCR) and sequenced: the internal transcribed spacer region (ITS) was generated using primers ITS1/ ITS4 (Munda 2015) and β-tubulin (TUB2) was amplified using primers Bt2a/Bt2b (Zhu et al. 2016). A BLAST analysis of the nucleotide sequence of the PCR products revealed 100% identity with two M. polystroma sequences in the NCBI GenBank (KJ814976 for ITS, KR778970 for TUB2). Our sequences were deposited in GenBank with accession nos. MT038413 for ITS and MT038414 for TUB2. On the basis of these results, the isolate was identified as M. polystroma. To confirm pathogenicity, 78 fresh and healthy bunches of ‘Beibinghong’ grapes at ripening were collected, surface disinfected by immersion in 1% NaOCl for 1 min, rinsed three times with sterile water, then allowed to air dry. Under dry aseptic conditions, the fruits were inoculated using the pin prick method. Each wound was inoculated with 10 μl conidial suspension (106 spore ml−1) and incubated at 25°C with about 90% relative humidity and natural light. Inoculation with water was used as control and the experiment was repeated three times. After a 10-day incubation, typical symptoms of brown rot developed on inoculated fruits, while control fruits were symptomless. The fungus was consistently re-isolated only from diseased fruits and showed the same morphological characteristics as the original isolates, thus fulfilling Koch’s postulates. This is the first report of M. polystroma on V. amurensis in China. The resulting disease decreases fruit quality and yield, necessitating the development of effective control measures.

The Search for Quorum Sensing in Botrytis cinerea: Regulatory Activity of Its Extracts on Its Development

Botrytis cinerea is a phytopathogenic fungus that causes large crop and post-harvest losses. Therefore, new and effective strategies are needed to control the disease and to reduce resistance to fungicides. Modulating pathogenicity and virulence by manipulating microbial communication is a promising strategy. This communication mechanism, called Quorum Sensing (QS), has already been reported in bacteria and yeasts; however, it has not yet been studied in B. cinerea. To establish the existence of this biochemical process in B. cinerea, we prepared extracts at different growth times (D1-D12), which were applied to fresh cultures of the same fungi. The chemical analysis of the extracts obtained from several fermentations showed different compositions and biological activities. We confirmed the presence of several phytotoxins, as well as compounds 1-phenylethanol and 3-phenylpropanol. Day five extract (0.1%) inhibited conidia germination and elongation of germ tubes, day seven extract (1%) produced the greatest phytotoxic effect in tomato leaves, and day nine extract (0.1%) was a sporulation inhibitor. In contrast, the extracts from days 7, 9, and 12 of fermentation (0.1% and 0.01%) promoted pellet and biofilm formation. Sporulation was slightly induced at 0.01%, while at 0.1% there was a great inhibition. At the highest extract concentrations, a biocidal effect was detected, but at the lowest, we observed a QS-like effect, regulating processes such as filamentation, morphogenesis, and pathogenesis. These results of the biological activity and composition of extracts suggest the existence of a QS-like mechanism in B. cinerea, which could lead to new non-biocidal alternatives for its control through interference in the pathogenicity and virulence mechanisms of the fungi.

Microfluidic platform for integrated compartmentalization of single zoospores, germination and measurement of protrusive force generated by germ tubes

We introduce a platform capable of trapping and compartmentalizing spores of oomycetes and fungi for germination and protrusive force sensing on individual germ tubes.