Pathogenic Fungi Diversity of ‘CuiXiang’ Kiwifruit Black Spot Disease during Storage

Kiwifruit black spot disease has become increasingly widespread in many ‘CuiXiang’ kiwifruit plantings regions. This research was aimed at the pathogenic microorganisms of black spot of the ‘CuiXiang’ cultivar. Physiological, morphological and transcriptional characteristics between black spot fruit and healthy fruits were evaluated. Then, it applied a high-throughput internal transcribed spacer (ITS) sequencing to analyze the black spot disease microbial community. The cell structure showed that mycelium was attached to the surface of the kiwifruit through black spot, and that consequently the mitochondria were damaged, starch particles were reduced, and shelf life was shortened. Transcriptome revealed that different genes in kiwifruit with black spot disease were involved in cell wall modification, pathogen perception, and signal transduction. ITS sequencing results described the disease-causing fungi and found that the microbial diversity of black spot-diseased fruit was lower than that of healthy fruit. We predict that candidate pathogenic fungi Cladosporium cladosporioides, Diaporthe phaseolorum, Alternaria alternata, and Trichothecium roseum may cause black spot. This study was to explore the pathogenic fungal community of ‘CuiXiang’ kiwifruit black spot disease and to provide essential information for field prevention.

The influence of black spot disease on the relative condition factor of Astyanax paranae Eigenmann, 1914 (Characiformes: Characidae) in Brazilian subtropical streams

Environmental disturbances and their consequences require constant studies to understand how communities and their ecological relationships respond to these processes. Through analysis of the host-parasite relationships, it is observed that the effect of these disturbances is variable and can change the physiology or behavior of organisms. Black spot disease, caused by endoparasitic helminths, is a pathology observed in natural environments, however, there is not much information about the consequences of this infestation. We separated the specimens from each stream into parasitized and non-parasitized groups, which were subjected to biometric analysis. The biometrics involved cysts count and weight-length measures, which were used to analyze the average relative condition factor. Additionally, we correlate these measures with the parasitic burden of infected individuals. Finally, the parasitized individuals were submitted to histological sections to recognize the parasite. The results demonstrate a low physiological condition in the parasitized group, when compared with non-parasitized groups from the same stream and from different streams. This suggests that pollution, in addition to effects of infestation worsen the fish condition. Besides, the parasite burden was negatively correlated with the condition factor, weight and length measures. We conclude that the parasite burden negatively affects Astyanax paranae individuals´ physiological condition and that trematodes also occur in polluted environments.

Alternaria Black Spot (Alternaria brassicae) Infection Severity on Cruciferous Oilseed Crops

The increase in the cultivation area of cruciferous oilseed crops and the use of short crop rotation has resulted in the enhanced spread of several major pests in Northern latitudes. There is currently limited information about incidence and severity of Alternaria black spot disease (Alternaria brassicae) on the main oilseed crop, spring oilseed rape (Brassica napus), in the Northern Baltics. Thus, spring oilseed rape and five alternative cruciferous oilseed crops were selected and their resistance to black spot disease was evaluated in field conditions during two growing seasons. We hypothesized that spring oilseed rape is more susceptible to Alternaria black spot disease than other alternative cruciferous oilseed crops. Both growing seasons were warmer and drier compared to the long-term average, and were thus suitable for A. brassicae development and assessments. In both years, incidence of Alternaria black spot infection was recorded on all cruciferous species, yet the disease development differed considerably among the crops. During both growing seasons, black mustard (B. nigra) plants were the most infected. Based on our observations during warm growing seasons we conclude that alternative oilseed crops such as Sinapis alba, Eruca sativa and Raphanus sativus are more resistant to the Alternaria black spot infection than the traditional oilseed crops and thus, possess a great potential to grow with limited chemical disease control in Northern Baltic conditions.

First report of Arcopilus aureus causing leaf black spot disease of Pseudostellaria heterophylla in China

Pseudostellaria heterophylla (family Caryophyllaceae) is a perennial herbaceous plant. Its tuberous roots are highly valued in traditional Chinese medicine. It is mainly cultivated in a geo-authentic production zone located in the Guizhou, Anhui, Shandong, and Fujian provinces of China (Zhao et al. 2016). The herb is widely used for treating lung diseases and as a spleen tonic (Pang et al. 2011). A severe leaf black spot disease was observed on P. heterophylla in China, from 2018 to 2020. Plants displayed water-soaking symptoms in the early stage of infection, then the watery areas turned brown-red and a black mold appeared on the lesions. At a later stage, the leaf spots showed concentric rings surrounded by a yellow halo, and the initial infection site became dry and necrotic (Supplementary Figure S1). Nine infected plants were collected from three cultivation fields in Shibing County (N 27°4'21", E 108°8'0"), Guizhou province, on April 13th, 2019. The fungus was consistently isolated from symptomatic leaves on potato dextrose agar (PDA) medium according to the method described by Larran et al (2002). A total of 22 isolates were obtained, including 7 isolates of Arcopilus and 15 isolates of Trichoderma. The growth rates of isolate MJ2-2b on PDA and oatmeal agar (OA) medium were 3 to 5 mm/day at 25 °C (Supplementary Figure S2A and S2B). Mycelium of isolate MJ2-2b was dense, yellowish-brown on PDA, while it was sparse, bright-red on OA. Also, the mycelium secreted brownish-red pigment on both PDA and OA. Ascomata when mature were water drop and limoniform. Lateral hairs were brown, erect or flexuous, tapering towards the tips. Ascospores when mature were greyish-white to grey, limoniform, or fusiform to pyriform (Supplementary Figure S2C and S2D). Further, the beta-tubulin gene (Tub2) of the fungus was amplified by using primer pairs T1 and TUB4Rd as described by Wang et al (2016) and subjected to sequencing. NCBI nucleotide BLAST results showed that sequences from seven isolates had a 99.86% identity with A. aureus (strain ChL-C, GenBank accession No. MG889987.1) (Supplementary Figure S2F). Molecular phylogenetic analysis by maximum likelihood method using MEGA 7 confirmed that the fungal isolate clustered with A. aureus. Hence, the causal agent was identified as A. aureus based on morphological and molecular characteristics. The sequence was deposited in GenBank (accession No. MW531453). Pathogenicity tests were conducted on 15-day old tissue-cultured seedlings according to Ghanbary et al (2018) (Supplementary Figure S3). Leaves of 16 seedlings were inoculated with 1×1 mm 5-day-old PDA-grown mycelial plugsof the fungal isolate. The experiment was repeated 3 times. After 10 days, the inoculated leaves showed the same symptoms observed on plants in the field. The associated fungal pathogen was consistently re-isolated from the inoculated seedlings and identified by Tub2 gene sequencing. At present, there are no reports of A. aureus causing disease of plants. To the best of our knowledge, this is the first report of leaf black spot disease on P. heterophylla caused by A. aureus in China.

Rapid and Specific Detection of the Poplar Black Spot Disease Caused by Marssonina brunnea Using Loop-Mediated Isothermal Amplification Assay

Marssonina brunnea is the main pathogen that causes poplar black spot disease, which leads to the decrease of the photosynthetic efficiency and significantly affects the production and quality of timber. Currently, no in-field diagnostic exists for M. brunnea. Here, we described a loop-mediated isothermal amplification (LAMP) assay for the rapid and sensitive detection of M. brunnea. A set of six oligonucleotide primers was designed to recognize eight distinct sequences of the internal transcribed spacer (ITS) region of M. brunnea. The LAMP assay was optimized by the combination of high specificity, sensitivity, and rapidity for the detection of less than 10 pg/μL of target genomic DNA in 60 min per reaction at 65 °C, whereas with PCR, there was no amplification of DNA with concentration less than 1 ng/μL. Among the genomic DNA of 20 fungalisolates, only the samples containing the genomic DNA of M. brunnea changed from violet to sky blue (visible to the naked eye) by using hydroxynaphthol blue (HNB) dye. No DNA was amplified from the eight other fungus species, including two other Marssonina pathogens, three other foliar fungi pathogens of poplar, and three common foliar fungal endophytes of poplar. Moreover, the detection rates of M. brunnea from artificially and naturally infected poplar leaves were 10/16 (62.5%) and 6/16 (37.5%) using PCR, respectively, while the positive-sample ratios were both 16/16 (100%) using the LAMP assay. Overall, the ITS LAMP assay established here can be a better alternative to PCR-based techniques for the specific and sensitive detection of M. brunnea in poplar endemic areas with resource-limited settings.