First report of Neocosmospora pisi causing blight on Ammopiptanthus mongolicus in China.

Ammopiptanthus mongolicus is the only evergreen broad-leaved shrub in the desert region of Northwest China, which is one of the dominant species in the desert vegetation of the region, playing an important role in maintaining the stability of the local desert ecosystem. A. mongolicus is also very hardy and drought resistant and can survive extreme temperatures (Liu et al. 2013; Yang et al. 2022). The large-scale death of A. mongolicus could cause desertification in the region. Two months after the discovery of Fusarium verticillioides causing blight on A. mongolicus in Etuoke county, Inner Mongolia Autonomous Region in September 2023 (Yang et al. 2024), a large number of A. mongolicus plants with symptoms of blights were found in Lingwu city, Ningxia Hui Autonomous Region, China (106.442368°E, 37.734026°N) in November 2023. The incidence of diseased plants in this field was about 30%. The field symptoms in Lingwu city were similar to those observed in Etuoke county. The diseased leaves initially turned yellow, then wilted and dehisced, eventually resulting in plant death (Figure 1). The roots of the diseased plants were cut diagonally and the central cylinder showed a brown color (Figure 2). In order to investigate whether the death of A. mongolicus was caused by the same pathogen as those identified previously, 30 roots were collected from 10 diseased plants. After rinsing and surface sterilization (70% ethanol for 3 min and 2.5% NaClO for 5 min, rinsed 3 times with sterile distilled water), diseased tissues (10×10 mm) were placed on potato dextrose agar (PDA) (3 pieces per plate) and incubated from 3 to 5 days at 25°C. The strain AmP5 was isolated and used for further study. After 3 days on PDA medium, fungal colonies were white to milky, the undersides of the cultures were yellowish to orange-brown (Figure 3). After 7 days on synthetic nutrient-poor agar (SNA), microconidia were ovoidal or with a rounded apex and truncate base, 10.5 ± 1.5 µm × 1.6 ± 0.2 µm (×400). The macroconidia were slightly curved or arcuate, 40.5 ± 3.5 µm × 5 ± 0.5 µm (×400) (Figure 4) (Sisic et al. 2018). The pathogen was confirmed to be Neocosmospora pisi by multigene phylogenetic analysis of TEF, RPB1 and RPB2 genes using primers EF1/EF2, F5/G2R and 5F2/11AR, respectively (O'Donnell et al. 2022). The sequences of PCR products were deposited in GenBank with accession numbers OR944631 (RPB1), OR988086 (TEF) and OR988087 (RPB2), respectively. The results of pairwise alignment in Fusarioid-ID database (Crous et al. 2021) showed 99.84% similarity and 83.96% overlap of the EF1-α sequence to the corresponding sequence LR583636 of ex-epitype CBS 123669 of Neocosmospora pisi (syn. Fusarium solani f. sp. pisi), 99.72% similarity and 85.66% overlap of the RPB1 sequence to the corresponding sequence MW834242 of ex-epitype CBS 123669 of N. pisi, and 99.47% similarity and 78.26% overlap of RPB2 sequence to the corresponding sequence LR583862 of ex-epitype CBS 123669 of N. pisi. Moreover, the result of polyphasic identification in the Fusarioid-ID database also showed EF1-a, RPB1, and RPB2 sequences had 99.15% similarity to the corresponding sequences of CBS 1233669. The pathogenicity of AmP5 was tested on potted 64 days old seedlings A. mongolicus plants. The roots of 3 seedlings were inoculated with conidial suspension (1×106 /ml), and another 3 used as controls were inoculated with sterile water, by gently peeling off the soil around the roots during inoculation, and pouring the conidial suspension around the roots (10 ml/seedling). All plants were placed in a growth chamber at 18-25℃ (10 h light; 14 h dark). After incubation for 3-5 days, the symptoms similar to those observed in the field (Figure 5), including brown rot of steles (Figure 6), developed on plants inoculated with conidial suspension, whereas no symptoms were observed on the control plants. The same pathogen was reisolated from inoculated roots and confirmed as N. pisi based on morphological and molecular analyses (TEF, RPB1 and RPB2). To our knowledge, this is the first report of blight on A. mongolicus caused by N. pisi in China. This study also indicates that blight on A. mongolicus can be caused by different fungal pathogens. Blight caused by different pathogens may have different in terms of control measures and pathogenic mechanisms, so the study of blight caused by different pathogens is of profound value.

First report of Fusarium verticillioides causing blight on Ammopiptanthus mongolicus in China.

Ammopiptanthus mongolicus is the only evergreen broadleaf shrub and constructive species in the northwest desert of China (Hu et al. 2021). It also is listed as one of the national second-class endangered plants. Ammopiptanthus mongolicus has a good effect of water conservation, windbreak and sand fixation because its deep root system (Zhou et al. 2012; Dong et al. 2023). A large number of dead plants of Ammopiptanthus mongolicus were found in Etuoke county, Inner Mongolia Autonomous Region, China (40°4'28″-40°4'34″ N, 106°53'5″-106°53'31″ E). In September 2023, the investigation and research in the region found that the incidence of diseased plants in this field was about 30%, and for individual plant, the incidence of diseased branches was about 60%. The leaves of diseased branches initially became from green to yellow and then wilt and fall. Eventually the plant dies. (Figure 1). The miter cut of the root showed that the root steles of diseased plants had obvious black and brown color (Figure 2). For isolation, the 30 tissue blocks (10×10 mm) of from 10 symptomatic roots diseased were surface sterilized with 70% ethanol for 3 minutes and sodium hypochlorite (2.5% available chlorine) for 5 minutes, and rinsed three times with sterilized distilled water. Then, these tissue blocks were placed on potato dextrose agar (PDA) medium, and incubated from 3 to 5 days at 25°C. After 3 days on PDA, the surface of the colony was rough, the color were white-pink at the beginning, and deep purple pigment were produced in the later stage, making the colony bluish-purple to gray-purple, their undersides were bluish-purple. Mycelia were white. After 7 days on SNA, Microconidia were typical of the clavicular type, 8.5 ± 2.5 µm × 2.3 ± 0.2 µm(×400). Microconidia were usually very long conidial chains, sometimes the spore chain collapses and the conidia clump together to form an approximate pseudocephaly. The macroconidia were slender and long, slightly falcate or straight, 42.8 ± 3.4 µm × 3.8 ± 0.7 µm(×400) (Figure 4). Species identity was confirmed by sequencing the EF1-α gene (EF1 and EF2 primers)(O'Donnell et al. 1998), RPB1 (F5 and G2R primers)(O'Donnell et al. 2022) and RPB2 (5F2 and 11AR)(O'Donnell et al. 2022). The amplified sequences of a representative isolate (AmP10) have deposited in GenBank with accession number OR594338 (EF1-α), OR841329 (RPB1) and OR841331 (RPB2). Thee results of pairwise alignment in Fusarioid-ID datebase(Crous et al. 2021) showed that EF1-α sequence was 99.54% similarity and 89.96% overlap to the corresponding sequence KF499582 of ex-epitype CBS 218.76 of Fusarium verticillioides, Fusarium fujikuroi species complex (FFSC, previously GFSC) (Lecellier et al. 2014), RPB1 sequence was 100% similarity and 100% overlap to the corresponding sequence MW402638 of ex-epitype CBS 218.76 of Fusarium verticillioides, Fusarium fujikuroi species complex (FFSC, previously GFSC) (Yilmaz et al. 2021), RPB2 sequence was 99.94% similarity and 87.83% overlap to the corresponding sequence MW928835 of ex-epitype CBS 218.76 of Fusarium verticillioides, Fusarium fujikuroi species complex (FFSC, previously GFSC) (Crous et al. 2021). Moreover, the result of polyphasic identification in Fusarioid-ID datebase also showed EF1-α, RPB1 and RPB2 sequences were 100% similarity to the corresponding sequences of ex-epitype CBS 218.76 of Fusarium verticillioides, Fusarium fujikuroi species complex (FFSC, previously GFSC). To test the pathogenicity, the healthy green seedlings (64 days old) were planted into plastic pots containing sterilized soil in the greenhouse after the seeds of Ammopiptanthus mongolicus were surface sterilized with 70% ethanol for 3 minutes and 2.5% sodium hypochlorite for 3 minutes. The roots of 3 seedlings were inoculated with 1×106 /ml of the conidial suspension, and another 3 used as controls with inoculated sterile water. Then, all pots were placed in a greenhouse maintained at 18°C to 25°C. After incubation for 3-5 days, the typical symptoms similar to the symptoms in the field (Figure 5), brown root steles (Figure 6), developed on the plants inoculated with conidial suspension, whereas no symptoms were observed on the control plants. The same pathogen was consistently reisolated from the inoculated roots and confirmed as Fusarium verticillioides based on morphological and molecular analyses. To our knowledge, this is the first report of Fusarium verticillioides on Ammopiptanthus mongolicus in China. This study provides a basis for identifying pathogens causing blight on Ammopiptanthus mongolicus and managing the disease.

Endophytic Fungi Regulate HbNHX1 Expression and Ion Balance in Hordeum bogdanii under Alkaline Stress.

Plants cope with abiotic stress in several ways, including by collaborating with microorganisms. Epichloë, an endophytic fungus, has been shown to improve plant tolerance to extreme external environments. Hordeum bogdanii is a known salt-tolerant plant with the potential to improve alkaline lands. NHX1 plays a key role in the transport of ions in the cell and is overexpressed in plants with increased salt tolerance. However, the expression levels of HbNHX1 in Epichloë endophytic fungal symbionts in H. bogdanii have not been elucidated. We used Hordeum bogdanii (E+) with the endophytic fungi Epichloë bromicola and H. bogdanii (E-) without the endophytic fungi and compared the differences in the ion content and HbNHX1 expression between the shoots and roots of E+ and E- plants under alkaline stress. The absorption capacity of both K+ and Na+ of H. bogdanii with endophytic fungi was higher than that without endophytic fungi. In the absence of alkaline stress, endophytic fungi significantly reduced the Cl- content in the host H. bogdanii. Alkaline stress reduced SO42- content in H. bogdanii; however, compared with E-, endophytic fungi increased the content of SO42- in E+ plants. With an increase in the alkaline concentration, the expression of HbNHX1 in the roots of H. bogdanii with endophytic fungus exhibited an upward trend, whereas the expression in the shoots exhibited a downward trend first and then an upward trend. Under 100 mmol·L-1 mixed alkaline stress, the expression of HbNHX1 in E+ was significantly higher than that in E-, indicating that endophytic fungi could increase the Na+ region in vacuoles. The external environment affects the regulation of endophytic fungi in H. bogdanii and that endophytic fungi can play a key role in soil salinization. Therefore, the findings of this study will provide technical support and a theoretical basis for better utilization of endophytic fungi from H. bogdanii in saline land improvement.

Effects of endophytic fungi on the secondary metabolites of Hordeum bogdanii under alkaline stress.

It is currently unclear whether the mechanism of endophytic fungi improving the alkali tolerance of Hordeum bogdanii affects secondary metabolites. Unveiling this knowledge is crucial for understanding the tolerance mechanism of H. bogdanii to alkaline stress. The aim of this study was to investigate how endophytic fungi affect secondary metabolites of H. bogdanii under alkaline stress at different concentrations. Endophyte-infected (E +) and endophyte-free (E-) individuals of H. bogdanii were used as materials in this study. The method of indoor vermiculite aseptic planting was adopted. After mixed alkali stress treatment, the roots, stems, and leaves of the plants were collected to measure the indicators related to secondary metabolites. The results showed that endophytic fungi improved the alkali resistance of H. bogdanii by improving the related indicators of secondary metabolites. endophytic fungi significantly increased the contents of phosphorus, polyphenols, and alkaloids, and the activities of polyphenol oxidase and acid phosphatase, and significantly reduced flavonoid content. The content of polyphenols and alkaloids in stems, polyphenol oxidase activity in stems and leaves, and acid phosphatase activity in leaves were significantly affected. The findings of this study may aid in amplifying the alkali resistance mechanism of endophytic fungi to H. bogdanii as well as provide insights into improving the alkali resistance of other plants.

Genome-Wide Analysis of Codon Usage Bias in Epichloë festucae.

Analysis of codon usage data has both practical and theoretical applications in understanding the basics of molecular biology. Differences in codon usage patterns among genes reflect variations in local base compositional biases and the intensity of natural selection. Recently, there have been several reports related to codon usage in fungi, but little is known about codon usage bias in Epichloë endophytes. The present study aimed to assess codon usage patterns and biases in 4870 sequences from Epichloë festucae, which may be helpful in revealing the constraint factors such as mutation or selection pressure and improving the bioreactor on the cloning, expression, and characterization of some special genes. The GC content with 56.41% is higher than the AT content (43.59%) in E. festucae. The results of neutrality and effective number of codons plot analyses showed that both mutational bias and natural selection play roles in shaping codon usage in this species. We found that gene length is strongly correlated with codon usage and may contribute to the codon usage patterns observed in genes. Nucleotide composition and gene expression levels also shape codon usage bias in E. festucae. E. festucae exhibits codon usage bias based on the relative synonymous codon usage (RSCU) values of 61 sense codons, with 25 codons showing an RSCU larger than 1. In addition, we identified 27 optimal codons that end in a G or C.