A highly effective and self-transmissible CRISPR antimicrobial for elimination of target plasmids without antibiotic selection

The use of CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein) for sequence-specific elimination of bacteria or resistance genes is a powerful tool for combating antibiotic resistance. However, this approach requires efficient delivery of CRISPR/Cas DNA cassette(s) into the targeted bacterial population. Compared to phage transduction, plasmid conjugation can deliver DNA to a broader host range but often suffers from low delivery efficiency. Here, we developed multi-plasmid conjugation systems for efficient CRISPR/Cas delivery, target DNA elimination and plasmid replacement. The CRISPR/Cas system, delivered via a broad-host-range R1162 mobilizable plasmid, specifically eliminated the targeted plasmid in recipient cells. A self-transmissible RK2 helper plasmid facilitated the spread of mobilizable CRISPR/Cas. The replacement of the target plasmid with another plasmid from the same compatibility group helped speed up target plasmid elimination especially when the target plasmid was also mobilizable. Together, we showed that up to 100% of target plasmid from the entire recipient population could be replaced even at a low (1:180) donor-to-recipient ratio and in the absence of transconjugant selection. Such an ability to modify genetic content of microbiota efficiently in the absence of selection will be critical for future development of CRISPR antimicrobials as well as genetic tools for in situ microbiome engineering.

Comparative Whole-Genome Sequence Analyses of Fusarium Wilt Pathogen (Foc R1, STR4 and TR4) Infecting Cavendish (AAA) Bananas in India, with a Special Emphasis on Pathogenicity Mechanisms

Fusarium wilt is caused by the fungus Fusarium oxysporum f. sp. cubense (Foc) and is the most serious disease affecting bananas (Musa spp.). The fungus is classified into Foc race 1 (R1), Foc race 2, and Foc race 4 based on host specificity. As the rate of spread and the ranges of the devastation of the Foc races exceed the centre of the banana’s origin, even in non-targeted cultivars, there is a possibility of variation in virulence-associated genes. Therefore, the present study investigates the genome assembly of Foc races that infect the Cavendish (AAA) banana group in India, specifically those of the vegetative compatibility group (VCG) 0124 (race 1), 0120 (subtropical race 4), and 01213/16 (tropical race 4). While comparing the general features of the genome sequences (e.g., RNAs, GO, SNPs, and InDels), the study also looked at transposable elements, phylogenetic relationships, and virulence-associated effector genes, and sought insights into race-specific molecular mechanisms of infection based on the presence of unique genes. The results of the analyses revealed variations in the organisation of genome assembly and virulence-associated genes, specifically secreted in xylem (SIX) genes, when compared to their respective reference genomes. The findings contributed to a better understanding of Indian Foc genomes, which will aid in the development of effective Fusarium wilt management techniques for various Foc VCGs in India and beyond.

Mycelial compatibility, anastomosis, and nucleus numbers of eight Mexican Hirsutella citriformis strains isolated from Diaphorina citri

Background Among entomopathogenic fungi, H. citriformis has been recognized as potential biocontrol agent against the Asian citrus psyllid Diaphorina citri (Hemiptera: Liviidae). Nevertheless, this fungus is poorly characterized. Previous molecular studies have shown high sequence similarities among strains, but significant differences in Diaphorina citri virulence. Objective The aim of the present study was to determine mycelial compatibility and anastomosis, and nucleus numbers in mycelium and conidia of eight H. citriformis strains isolated from mycosed D. citri adults collected from several Mexican states. Methods Mycelial compatibility and anastomosis evaluation was performed after pairing strains, leading to 36 confrontations, and cultured in chlorate minimum medium to obtain mutants for vegetative compatibility group. Results Hypha or conidia nuclei were visualized with safranin-O and 3% KOH, and 0.05% trypan blue–lactophenol solution. H. citriformis strains showed compatibly and anastomosis events after confrontation. In addition, they showed one nucleus per conidium and mycelium section. It was not possible to obtain H. citriformis nit mutants from the chlorate concentrations tested. Conclusions To date, this is the first report demonstrating mycelial compatibility, anastomosis occurrence, and hyphae and conidia nuclei number among H. citriformis strains.

Draft Genome of Fusarium oxysporum f. sp. cubense Strain Tropical Race-4 Infecting Cavendish (AAA) Group of Banana in India

Fusarium wilt, caused by the fungus Fusarium oxysporum f. sp. cubense, is the most serious pandemic disease of banana. In this study, we report the draft genome of F. oxysporum f. sp. cubense vegetative compatibility group (VCG) 01213/16 of strain tropical race 4 (TR4) that infects the Cavendish (AAA) group of banana collected from the subtropical region in India. The genome assembly of SFoc TR4 comprises 47,384,463 bp with 4,034 contigs and 15,508 protein-coding regions. Based on VCG analysis, the fungal isolate belongs to F. oxysporum f. sp. cubense TR4 but the genome sequence of SFoc TR4 shows differences in secreted-in-xylem (SIX) protein gene clusters (specifically, SIX8) in comparison with the reference genome of F. oxysporum f. sp. lycopersici and F. oxysporum f. sp. cubense TR4.

Genomic differences between the new Fusarium oxysporum f. sp. apii (Foa) race 4 on celery, the less virulent Foa races 2 and 3, and the avirulent on celery f. sp. coriandrii

Background Members of the F. oxysporium species complex (FOSC) in the f. sp. apii (Foa) are pathogenic on celery and those in f. sp. coriandrii (Foci) are pathogenic on coriander (=cilantro). Foci was first reported in California in 2005; a new and highly aggressive race 4 of Foa was observed in 2013 in California. Preliminary evidence indicated that Foa can also cause disease on coriander, albeit are less virulent than Foci. Comparative genomics was used to investigate the evolutionary relationships between Foa race 4, Foa race 3, and the Foci, which are all in FOSC Clade 2, and Foa race 2, which is in FOSC Clade 3. Results A phylogenetic analysis of 2718 single-copy conserved genes and mitochondrial DNA sequence indicated that Foa races 3 and 4 and the Foci are monophyletic within FOSC Clade 2; these strains also are in a single somatic compatibility group. However, in the accessory genomes, the Foci versus Foa races 3 and 4 differ in multiple contigs. Based on significantly increased expression of Foa race 4 genes in planta vs. in vitro, we identified 23 putative effectors and 13 possible pathogenicity factors. PCR primers for diagnosis of either Foa race 2 or 4 and the Foci were identified. Finally, mixtures of conidia that were pre-stained with different fluorochromes indicated that Foa race 4 formed conidial anastomosis tubes (CATs) with Foci. Foa race 4 and Foa race 2, which are in different somatic compatibility groups, did not form CATs with each other. Conclusions There was no evidence that Foa race 2 was involved in the recent evolution of Foa race 4; Foa race 2 and 4 are CAT-incompatible. Although Foa races 3 and 4 and the Foci are closely related, there is no evidence that either Foci contributed to the evolution of Foa race 4, or that Foa race 4 was the recent recipient of a multi-gene chromosomal segment from another strain. However, horizontal chromosome transfer could account for the major difference in the accessory genomes of Foa race 4 and the Foci and for their differences in host range.

Genetic Diversity of Fusarium oxysporum f. sp. cubense, the Fusarium Wilt Pathogen of Banana, in Ecuador

The continued dispersal of Fusarium oxysporum f. sp. cubense Tropical race 4 (FocTR4), a quarantine soil-borne pathogen that kills banana, has placed this worldwide industry on alert and triggered enormous pressure on National Plant Protection (NPOs) agencies to limit new incursions. Accordingly, biosecurity plays an important role while long-term control strategies are developed. Aiming to strengthen the contingency response plan of Ecuador against FocTR4, a population biology study—including phylogenetics, mating type, vegetative compatibility group (VCG), and pathogenicity testing—was performed on isolates affecting local bananas, presumably associated with race 1 of F. oxysporum f. sp. cubense (Foc). Our results revealed that Foc populations in Ecuador comprise a single clonal lineage, associated with VCG0120. The lack of diversity observed in Foc populations is consistent with a single introduction event from which secondary outbreaks originated. The predominance of VCG0120, together with previous reports of its presence in Latin America countries, suggests this group as the main cause of the devastating Fusarium wilt epidemics that occurred in the 1950s associated to the demise of ‘Gros Michel’ bananas in the region. The isolates sampled from Ecuador caused disease in cultivars that are susceptible to races 1 and 2 under greenhouse experiments, although Fusarium wilt symptoms in the field were only found in ‘Gros Michel’. Isolates belonging to the same VCG0120 have historically caused disease on Cavendish cultivars in the subtropics. Overall, this study shows how Foc can be easily dispersed to other areas if restriction of contaminated materials is not well enforced. We highlight the need of major efforts on awareness and monitoring campaigns to analyze suspected cases and to contain potential first introduction events of FocTR4 in Ecuador.

The Population of Fusarium oxysporum f. sp. lactucae in California and Arizona

Fusarium wilt of lettuce, caused by Fusarium oxysporum f. sp. lactucae, is now found in all major lettuce producing regions in California and Arizona. The population structure of F. oxysporum f. sp. lactucae in California and Arizona was characterized based on somatic compatibility and sequences of the translation elongation factor 1-α gene (EF-1α) and rDNA intergenic spacer region (IGS). In this study, 170 isolates were tested for somatic compatibility based on heterokaryon formation, using complementary nitrate nonutilizing (nit) mutants. Five subgroups (A to E) of somatic compatibility group 0300 were identified. Isolates associated with the same subgroup had a strong complementation reaction, whereas reactions between isolates of different subgroups were weak or delayed. An isolate from the first known infestation of Fusarium wilt of lettuce in California was associated with subgroup A, which predominated among isolates in our collection. Isolates representative of each subgroup were confirmed to be associated with race 1, based on the reaction of differential lettuce cultivars. It is possible that somatic compatibility subgroups B to E of F. oxysporum f. sp. lactucae were derived from subgroup A, as a consequence of somatic mutations affecting compatibility. If so, subgroups of F. oxysporum f. sp. lactucae may represent an intermediate step in divergence that will lead to clearly separable compatibility groups. Sequences of EF-1α and IGS were both identical for 58 isolates of F. oxysporum f. sp. lactucae that represented all somatic compatibility subgroups and locations from which isolates were obtained, indicating that subgroups were derived from the same clonal lineage (VCG 0300).

Training With Direct Versus Indirect Spatial Stimulus–Response Compatibility in Combat Sports

The study examined effects of spatial stimulus–response compatibility on response time and response accuracy in 20 novice combat sport athletes. Two equivalent groups, based on initial reaction time measures, were required to perceive and move quickly and accurately in response to an unspecific visual stimulus presented on a large screen during the two types of perceptual training in eight laboratory sessions. One group reacted by moving the fist toward the stimulus location on the target (direct compatibility condition). Another group was required to move the fist away from target in the opposite direction (indirect compatibility condition). Specifically, the indirect compatibility group achieved faster reaction times than the direct compatibility group during the two posttests containing video-projected attacks of the opponents, and in one of the two posttests containing real opponents’ attacks. Results seem to reveal higher combat performance against real opponents when athletes trained with an indirect stimulus–response compatibility condition.

Virulence not linked with vegetative compatibility groups in Australian cotton Verticillium dahliae isolates

Verticillium dahliae, the causal agent of Verticillium wilt, is a soil-borne ascomycete that infects numerous agriculturally important crops globally, including cotton. As a billion-dollar industry, cotton is economically important to Australia and the management of disease such as Verticillium wilt is key for the success of the industry. Internationally, defoliating V. dahliae isolates belonging to Vegetative Compatibility Group (VCG) 1A cause severe damage to cotton, while non-defoliating VCG2A isolates result in significantly less disease. However, in Australia, VCG2A is causing more severe damage to crops in the field than the defoliating VCG1A. This study aimed to replicate field observations in controlled greenhouse conditions. We examined and compared disease symptoms on a range of Australian commercial cotton varieties when inoculated with different V. dahliae VCGs. Seedlings were root dipped in conidial suspensions and assessed over seven weeks. The final disease score, disease over time and root length were analysed. Plant mortality resulted from both V. dahliae VCG1A and VCG2A isolates across all cotton varieties used, confirming that there are virulent VCG2A isolates present in Australia. To our knowledge, although virulent on other plant hosts, V. dahliae VCG2A has not previously been reported to be highly virulent in cotton. We infer that virulence cannot be defined solely by VCG in Australian V. dahliae isolates causing disease in cotton.