First Report of Fusarium falciforme (FSSC 3+4) Causing Rot of Industrial Hemp (Cannabis sativa) in California

Industrial hemp (Cannabis sativa) is a newly legal crop in California that is grown for cannabidiol oil, fiber and seed. In August 2019, whole plant decline and root rot were observed affecting <5% of plants in two industrial fields in Fresno County, CA. Symptoms included chlorotic, collapsed foliage, stem vascular discoloration, and root rot with abundant mycelial growth. Stem and root segments (1-2 cm) from three to five diseased plants were agitated in 0.1% tween-20 and soaked in 70% ethanol for 30 s and 1% NaOCl for 2 min. After incubating for 5 to 7 days on 1:10 potato dextrose agar (PDA) amended with tetracycline, Fusarium selective medium (FSM), and PARP (pimaricin + ampicillin + rifampicin + pentachloronitrobenzene [PCNB] agar) medium, white to pale cream aerial mycelium emerged from tissue of all plants on PDA and FSM but not PARP. Isolates cultured on 0.1% potassium chloride agar formed heads of microconidia on long monophialides consistent with the Fusarium solani species complex (FSSC) (Leslie and Summerell 2008). To obtain pure cultures of two isolates (CS529 and CS530), a single-hyphal tip was excised and grown on PDA. DNA was extracted from actively growing mycelium (PrepMan Ultra kit). The translation elongation factor gene (EF-1α) was amplified via PCR using EF1/EF2 primers (O’Donnell et al. 1998). Sequences of the two isolates were identical and deposited under accession number MW892973 in GenBank. The 599 bp sequence was 99.33% identical to FSSC 3 + 4 (Fusarium falciforme) accessions FD_01443_EF-1a based on FUSARIUM-ID BLAST analysis. To evaluate pathogenicity, stems of hemp plants (cv. ‘Berry Blossom’; n=8 plants per isolate) were wounded by penetrating the epidermis in an area about 0.5-cm square by 1-mm deep and 8-inches above the soil line. A 0.5 cm-diameter plug of 7-day old F. falciforme-colonized PDA was placed against the wound. Inoculation sites were loosely wrapped with parafilm for 2 days. A negative control consisted of a sterile PDA plug (n=3). Treatments were arranged in a completely randomized design in a greenhouse. The experiment was conducted once, due to regulatory restrictions at campus facilities. At 61 days post-inoculation, external stem lesions were significantly larger in diameter (P < 0.05; Tukey’s HSD) in plants inoculated with CS529 (8 ± 1 mm) compared to the control (2 ± 0 mm), and larger but not significant for CS530 (6 ± 1 mm). Internal stem lesions (i.e., rot in stele) were observed in plants inoculated with CS529 (9 ± 3 mm); stem rot was very minor in plants treated with CS530 (1 ± 1 mm) and nonexistent for control plants. No other disease symptoms were observed. F. falciforme was isolated from stems of CS529- and C530-inoculated plants. Sequences of re-isolates matched 100% with accession MW892973. These results suggest that F. falciforme causes rot in hemp in California. These studies specifically confirm stem rot abilities; field observations of root rot indicate root rotting abilities, but further tests are needed for confirmation. This is the first report of F. falciforme causing disease in industrial hemp. FSSC was described as causing foot rot in hemp in Italy (Sorrentino et al. 2019), but these isolates belonged to phylogenetic species 5 (F. solani) not F. falciforme. In addition, F. falciforme was reported as causing root rot in hydroponically grown cannabis (Punja and Rodriguez 2018). These studies provide the foundation for development of management tools for hemp disease.

Evidence for the Widespread Occurrence of Bacteria Implicated in Acute Oak Decline from Incidental Genetic Sampling

Acute Oak Decline (AOD) is complex syndrome affecting Britain’s keystone native oak species, (Quercus robur L. and Q. petraea L. (Matt.) Liebl.), in some cases causing mortality within five years of symptom development. The most distinguishable symptom is weeping stem lesions, from which four species of bacteria have been isolated: Brenneria goodwinii, Gibbsiella quercinecans, Lonsdalea britannica and Rahnella victoriana. We do not yet know where else these bacteria exist, and little is known about the relationship of the wider oak leaf microbiome (phyllosphere) to acute oak decline. Here we investigate whether incidental evidence from a large oak genome re-sequencing dataset could be used to detect these bacteria in oak foliage, and whether bacterial incidence co-varied with AOD status or location. Oak leaves and buds were sampled from 421 trees at five sites in England. Whole genomic DNA from these samples was shot-gun sequenced with short reads. Non-oak reads were extracted from these data and queried to microbial databases. Reads uniquely matching AOD-associated bacterial genomes were found to be present on trees from all five sites and included trees with active lesions, trees with historic lesions and trees without AOD symptoms. The abundance of the AOD-associated bacteria did not differ between tree health categories but did differ among sites. We conclude that the AOD-associated bacteria may be members of the normal oak microbiome, whose presence on a tree is not sufficient to cause AOD symptoms.

Evidence for the Widespread Occurrence of Bacteria Implicated in Acute Oak Decline from Incidental Genetic Sampling

Acute Oak Decline (AOD) is complex syndrome affecting Britain&rsquo;s keystone native oak species, (Quercus robur L. and Q. petraea L. (Matt.) Liebl.), in some cases causing mortality within five years of symptom development. The most distinguishable symptom is weeping stem lesions, from which four species of bacteria have been isolated: Brenneria goodwinii, Gibbsiella quercinecans, Lonsdalea britannica and Rahnella victoriana. We do not yet know where else these bacteria exist, and little is known about the relationship of the wider oak leaf microbiome (phyllosphere) to acute oak decline. Here we investigate whether incidental evidence from a large oak genome re-sequencing dataset could be used to detect these bacteria in oak foliage, and whether bacterial incidence co-varied with AOD status or location. Oak leaves and buds were sampled from 421 trees at five sites in England. Whole genomic DNA from these samples was shot-gun sequenced with short reads. Non-oak reads were extracted from these data and queried to microbial databases. Reads uniquely matching AOD-associated bacterial genomes were found to be present on trees from all five sites and included trees with active lesions, trees with historic lesions and trees without AOD symptoms. The abundance of the AOD-associated bacteria did not differ between tree health categories but did differ among sites. We conclude that the AOD-associated bacteria may be members of the normal oak microbiome, whose presence on a tree is not sufficient to cause AOD symptoms.

Phytophthora heterospora sp. nov., a New Pseudoconidia-Producing Sister Species of P. palmivora

Since 1999, an unusual Phytophthora species has repeatedly been found associated with stem lesions and root and collar rot on young olive trees in Southern Italy. In all cases, this species was obtained from recently established commercial plantations or from nursery plants. Morphologically, the Phytophthora isolates were characterized by the abundant production of caducous non-papillate conidia-like sporangia (pseudoconidia) and caducous papillate sporangia with a short pedicel, resembling P. palmivora var. heterocystica. Additional isolates with similar features were obtained from nursery plants of Ziziphus spina-christi in Iran, Juniperus oxycedrus and Capparis spinosa in Italy, and mature trees in commercial farms of Durio zibethinus in Vietnam. In this study, morphology, breeding system and growth characteristics of these Phytophthora isolates with peculiar features were examined, and combined mitochondrial and nuclear multigene phylogenetic analyses were performed. The proportion between pseudoconidia and sporangia varied amongst isolates and depended on the availability of free water. Oogonia with amphigynous antheridia and aplerotic oospores were produced in dual cultures with an A2 mating type strain of P. palmivora, indicating all isolates were A1 mating type. Phylogenetically, these isolates grouped in a distinct well-supported clade sister to P. palmivora; thus, they constitute a separate taxon. The new species, described here as Phytophthora heterospora sp. nov., proved to be highly pathogenic to both olive and durian plants in stem inoculation tests.

Effect of Different Botanical Extracts and Organic Compound in The Management of Leaf Spot Disease of Strawberry (Fragaria ananassa Duch.) Caused by Pestalotia longisetula under Field Condition

A field experiment was carried out using sweet sensation variety of strawberry to check the efficacy of different botanical extracts and organic compounds to manage leaf spot disease. The study was done using ten treatments viz; ginger (T1), turmeric (T2), garlic (T3), ginger + turmeric + garlic (T4), cow urine (T5), cow urine+ ginger + turmeric + garlic (T6), compost tea (T7), compost tea+ ginger+ turmeric+ garlic (T8), SAAF (mancozeb 63 % + carbendazim 12%) (T9) and control (T10), which was replicated thrice. The parameter observed during experiment were plant height, leaf number, disease leaves, stem lesions, yield, percent disease incidence, disease severity percent and percent disease control. At 75 days of transplantation, the highest plant height, leaf number (24.60) and yield (1391.67 gm/plot) were observed in treatment compost tea (18.14 cm), cow urine+ ginger+ turmeric+ garlic and garlic respectively and lowest in treatment control (16.19 cm, 23.27 & 566.67 gm/plot). The disease leaves and stem lesions were observed highest in treatment control (3.40&3.23) and lowest in treatments garlic (3.0), and cow urine+ ginger+ turmeric+ garlic (1.83) respectively at 75 days after transplantation. Moreover, the highest percent disease incidence and disease severity percent were observed highest in treatments control (28.94% & 84.81%) and percent disease control in treatment garlic (42.36%) at 75 days of transplantation. Therefore, it is suggested to use garlic extracts as a measure to control leaf spot disease of strawberry. Int. J. Appl. Sci. Biotechnol. Vol 9(3): 193-202.

Mangrove Dieback and Leaf Disease in Sonneratia apetala and Sonneratia caseolaris in Vietnam

Even though survival rates for mangrove restoration in Vietnam have often been low, there is no information on fungal pathogens associated with mangrove decline in Vietnam. Therefore, this research was undertaken to assess the overall health of mangrove afforestation in Thanh Hoa Province and fungal pathogens associated with tree decline. From a survey of 4800 Sonneratia trees, the incidence of disorders was in the order of pink leaf spot > shoot dieback > black leaf spot for S. caseolaris and black leaf spot > shoot dieback > pink leaf spot for S. apetala. Approximately 12% of S. caseolaris trees had both pink leaf spot and shoot dieback, while only 2% of S. apetala trees had black leaf spot and shoot dieback. Stem and leaf samples were taken from symptomatic trees and fungi were cultured in vitro. From ITS4 and ITS5 analysis, four main fungal genera causing leaf spots and shoot dieback on the two Sonneratia species were identified. The most frequently isolated fungal taxa were Curvularia aff. tsudae (from black leaf spot),Neopestalotiopsis sp.1 (from stem dieback), Pestalotiopsis sp.1 (from pink leaf spot), and Pestalotiopsis sp.4a (from black leaf spot). The pathogenicity of the four isolates was assessed by under-bark inoculation of S. apetala and S. caseolaris seedlings in a nursery in Thai Binh Province. All isolates caused stem lesions, and Neopestalotiopsis sp.1 was the most pathogenic. Thus, investigation of fungal pathogens and their impact on mangrove health should be extended to other afforestation projects in the region, and options for disease management need to be developed for mangrove nurseries.

Occurrence of root rot caused by Pythium aphanidermatum on mung bean (Vigna radiata) in China

In Aug 2019, approximately 10% of mung bean plants at the experimental farm of the Jiangsu Academy of Agricultural Science (32.03 N; 118.88 E) showed symptoms of stunting and wilting. Brown and water-soaked stem lesions were often observed at the base of the diseased plants. In severe cases, the plants collapsed and cumulous aerial mycelia were visible on the basal stem surface (Figure S1 A). To identify the causal agent, a total of 20 tissue fragments (5 mm long) were excised from roots and basal stems of five symptomatic plants. The fragments were surface sterilized in 2% sodium hypochlorite solution then plated on 2.5% potato dextrose agar (PDA) plates containing 10 μg/mL pimaricin, 100 μg/mL ampicillin, 10 μg/mL rifampicin, and 10 μg/mL pentachloronitrobenzene (PARP; Beckerman et al. 2017). After 3-4 days incubation at 25oC in dark, 14 colonies with white and cumulous mycelia grew from the tissue pieces (named as JS19-1 to JS19-14). JS19-1 and JS19-2 were purified by hyphal tipping, then grown on PDA medium for 7 days for morphological observation using a compound microscope (Figure S1 B, C). Width of coenocytic hyphae ranged from 3.7 to 8.9 (avg. 6.1, n=20) μm. Terminal oogonia were globose and with a diameter of 13.8 to 25.8 (avg. 22, n=20) μm. Antheridia were barrel-shaped, and mostly intercalary, sometimes terminal. Most of antheridia were diclinous, with 6.2 to 12.5 (avg. 9.3, n=20) μm in width and 7.6 to 15.3 (avg. 12.8, n=20) μm in length. Oogonia were fertilized with one or two (rare) antheridia. Oospores were aplerotic, 10.1 to 23.5 (avg. 20.4, n=20) μm in diameter. Sporangia had terminal inflated hyphal branches (Figure S1 D, E). The two isolates were preliminary identified as Pythium aphanidermatum. For molecular identification, the sequences of internal transcribed spacer (ITS) rDNA, cytochrome oxidase subunit I (CoxI) (Robideau et al. 2011), and β-tubulin (Kroon et al. 2004) of JS19-1 were detected, and deposited in GenBank (MT949538, MT949539 and MT949540). The ITS and CoxI sequences were identical with P. aphanidermatum CBS28779 ITS (759/759 bp, HQ643439.1) and PYT01 CoxI (640/640 bp, MH760243.1) respectively, the β-tubulin sequence showed 99% (830/840 bp) similarity of P. aphanidermatum P2 (AY564048.1). Thus, JS19-1 was confirmed as P. aphanidermatum. To fulfill Koch’s postulates, the pathogenicity of JS19-1 was tested using the procedure of Kiyoshi et al. (2021) with some modifications. Barley grains infested with JS19-1 were as inoculum and thoroughly mixed with potting mixture at a rate of 10% in volume. Six mung bean seeds were sown per pot and then grown in a greenhouse. Potting mixture with no inoculum was used as control. Three pots of replicate plants used for inoculation and control. After 3 weeks, emergence in the inoculated pots was 33% and symptoms of stunting and root rot similar to those in field were observed, while control plants were asymptomatic (FigureS1 F, G). P. aphanidermatum was successfully reisolated from symptomatic plants of both methods. The pathogenicity tests were repeated twice. P. aphanidermatum causes seed rot, pre- and postemergence damping-off, or stem/root rot of a wide range of industrial crops and vegetables (Liu et al, 2018). To our knowledge, this is the first report of P. aphanidermatum causing disease on mung bean in China. Since Phytophthora vignae (Sun et al, 2020) and P. myriotylum (Yan et al, 2021) have been reported causing mung bean root rot, integrated disease management should be adopted to reduce damage.

Improved Detectability of Brain Stem Ischemia by Combining Axial and Coronal Diffusion-Weighted Imaging

Background and Purpose: To evaluate the benefit of a coronal diffusion-weighted imaging (DWI) in addition to standard axial DWI for the detection of brain stem infarctions. Methods: A retrospective analysis of patients with symptoms consistent with acute and subacute brain stem infarction who received magnetic resonance imaging, including axial and coronal DWI. Diffusion restrictions were identified by 2 independent raters blinded for the final clinical diagnosis in 3 separate reading steps: axial DWI, coronal DWI, and combined axial and coronal DWI. Lesion location and certainty level were both documented for each reading step. In cases of reader disagreement, an additional consensus reading was performed. Results: Two hundred thirty-nine patients were included. Of these, 124 patients (51.9%) were clinically diagnosed with brain stem infarction. Sensitivity, specificity, positive, and negative predictive values were best for combined DWI assessment (90.3%, 99.1%, 99.1%, and 90.5%) compared with axial (85.5%, 94.9%, 94.6%, and 85.8%) and coronal DWI alone (87.9%, 96.5%, 96.5%, and 88.1%). Diffusion restriction on combined DWI was diagnosed in 112/124 patients compared with 106/124 on axial DWI and 109/124 on coronal DWI. Interobserver agreement for the detection of brain stem lesions was the highest in the combined rating step (Cohen κ coefficient=0.94). Conclusions: Coronal DWI sequences might improve the detection rate of brain stem infarction compared with standard axial DWI. The combined coronal and axial DWI provides the best detection rate while minimally increasing scan times.

Phomopsis vexans (Phomopsis blight of eggplant).

P. vexans is a pynicidial anamorph with a teleomorph in the genus Diaporthe. Easily seedborne and producing large numbers of conidia, it causes disease in Solanum melongena [aubergine/brinjal/eggplant], its only significant host. This ranges from poor seed germination and damping-off of seedlings, to leaf and stem lesions and to fruit rot, both in the field and after harvest. The fungus has been reported from widely distributed areas of most continents, but only a few of those are in Europe and Africa, even though the climates are favourable. Seed transmission may explain its broad historical distribution, but limitation of its host range to a non-staple vegetable crop can allow for its avoidance and eradication by cultural methods. As a result, perhaps, it does not appear often on lists of restricted pathogens, even though it may cause yield losses of more than 50%.