Serendipitous identification of a new Iflavirus-like virus infecting tomato and its subsequent characterization

The genomic sequence of a previously undescribed virus was identified from symptomless tomato plants (Solanum lycopersicum). The viral genome is a positive-sense ssRNA molecule of 8506 nucleotides. It is predicted to encode a single polyprotein of 314·5 kDa that is subsequently processed into three coat protein components of 13·7, 17·9 and 13·5 kDa, and a viral replicase of approximately 207 kDa with conserved motifs for a helicase, a protease and RNA-dependent RNA polymerase (RdRp). Pairwise analysis of the deduced amino acid sequence of the RdRp revealed that it shares closest identity with members of the family Iflaviridae, genus Iflavirus (19–47% identity). Evidence of replication in plants was detected by RT-PCR of the viral replicative strand, and short interfering RNAs (siRNAs) matching the virus. The name Tomato matilda virus (TMaV) is proposed, and furthermore, that the genus Tomavirus (Tomato matilda virus) be created within the family Iflaviridae. This is the first report of a plant-infecting virus resembling members of the Iflaviridae

Attempt to silence genes of the RNAi pathways of the root-knot nematode, Meloidogyne incognita results in diverse responses including increase and no change in expression of some genes

Control of plant-parasitic nematodes (PPNs) via host-induced gene silencing (HIGS) involves rational selection of genes and detailed assessment of effects of a possible knockdown on the nematode. Some genes by nature may be very important for the survival of the nematode that knockdown may be resisted. Possible silencing and effects of 20 such genes involved in the RNA interference (RNAi) pathways of Meloidogyne incognita were investigated in this study using long double-stranded RNAs (dsRNAs) as triggers. Two of the genes, ego-1 and mes-2, could not be knocked down. Expression of six genes (xpo-1, pash-1, xpo-2, rha-1, ekl-4, and csr-1) were significantly upregulated after RNAi treatment whereas for 12 of the genes, significant knockdown was achieved and with the exception of mes-2 and mes-6, RNAi was accompanied by defective phenotypes in treated nematodes including various degrees of paralysis and abnormal behaviors and movement such as curling, extreme wavy movements, and twitching. These abnormalities resulted in up to 75% reduction in infectivity of a tomato host, the most affected being the J2s previously treated with dsRNA of the gfl-1 gene. For 10 of the genes, effects of silencing in the J2s persisted as the adult females isolated from galls were under-developed, elongated, and transparent compared to the normal saccate, white adult females. Following RNAi of ego-1, smg-2, smg-6, and eri-1, reduced expression and/or the immediate visible effects on the J2s were not permanent as the nematodes infected and developed normally in tomato hosts. Equally intriguing was the results of RNAi of the mes-2 gene where the insignificant change in gene expression and behavior of treated J2s did not mean the nematodes were not affected as they were less effective in infecting host plants. Attempt to silence drsh-1, mut-7, drh-3, rha-1, pash-1, and vig-1 through HIGS led to reduction in nematode infestation by up to 89%. Our results show that genes may respond to RNAi knockdown differently so an exhaustive assessment of target genes as targets for nematode control via RNAi is imperative

Differing requirements for flavonoids during the formation of lateral roots, nodules and root knot nematode galls in Medicago truncatula

In this study, we tested whether the organogenesis of symbiotic root nodules, lateral roots and root galls induced by parasitic root knot nematodes (Meloidogyne javanica) was regulated by the presence of flavonoids in the roots of Medicago truncatula. Flavonoids accumulate in all three types of root organ, and have been hypothesized previously to be required for secondary root organogenesis because of their potential role as auxin transport regulators. • Using RNA interference to silence the flavonoid biosynthetic pathway in M. truncatula, we generated transformed flavonoid-deficient hairy roots which were used to study flavonoid accumulation, cell division and organogenesis of nodules, lateral roots and root galls. • Flavonoid-deficient roots did not form nodules, as demonstrated previously, but showed altered root growth in response to rhizobia. By contrast, flavonoid-deficient roots showed no difference in the number of lateral roots and root galls. Galls on flavonoid-deficient roots formed normal giant cells, but were shorter, and were characterized by reduced numbers of dividing pericycle cells. • We rejected the hypothesis that flavonoids are required as general regulators of the organogenesis of secondary root organs, but flavonoids appear to be necessary for nodulation. Possible reasons for this difference in the requirement for flavonoids are discussed

Comparisons between sequenced and re-sequenced genomes of historical subterranean clover mottle virus isolates

We report comparisons between the complete genomic sequences of five historical Western Australian isolates of subterranean clover mottle virus (SCMoV) from 1989–2000, and an infectious clone of its 1989 isolate. Sanger Sequencing (SS) and High Throughput Sequencing (HTS), or both, were used to obtain these genomes. Four of the SCMoV isolates were sequenced by SS in 1999–2002, but re-sequenced again by HTS in 2020. The pairs of sequences obtained from these four isolates differed by only 18–59 nucleotides. This small difference resulted from the different sequencing methods, the < 1–5 years each isolate was host passaged before freeze-drying prior to HTS sequencing, or a combination of both. Since SCMoV has not been reported outside Australia, this similarity suggests the population sequenced represents the progeny of either an indigenous virus that spread from a native legume to subterranean clover after its introduction or a recent seed-borne incursion from elsewhere. The ORF1 was the most variable, and the phylogenetic tree constructed with ORF1s showed the isolates grouped according to their symptom severity in subterranean clover, indicating the probability that ORF1-encoded P1 protein is a symptom determinant. A satellite RNA was associated with all SCMoV genomes obtained by HTS but none derived by SS

Nematode-induced syncytium - A multinucleate transfer cell

The formation and structure of a syncytium induced by the potato cyst-nematode (Heterodera rostochiensis Woll.) in potato roots is described. At the permanent feeding site of the nematode larva, usually in the root cortex, the larva pierces a cell with its mouth stylet and injects saliva. Cell wall dissolution occurs to incorporate neighbouring cells into a syncytium. A column of cells is incorporated towards the vascular tissue. Centripetal advance is limited by the lignifled xylem, then syncytial spread continues laterally along xylem parenchyma and pericycle cells. Wall protuberances form on syncytial walls adjacent to conducting elements. This indicates the syncytium is a multinucleate transfer cell, and by ingesting syncytial contents the larva is the nutrient sink. As syncytial expansion occurs, sieve elements are crushed and probably cease to function, hence protuberance development continues only against xylem elements. Cell alterations on incorporation into the syncytium involve expansion, loss of cell vacuole, nuclear hypertrophy and a proliferation of cytoplasmic organelles free to move through wall gaps into the communal cytoplasm. ‘Boundary formations’ and microtubules are associated with the growing ends of protuber ances, and appear to be involved in their synthesis. Fibrillar material, possibly cellulose microfibrils, occurs between the plasrnalemma and the membrane of the ‘boundary formation’, and the forming protuberance. To induce the formation of the syncytium, the larva controls the differentiation of unspecialized cells to cells with a specific physiological function. The occurrence of wall protuberances suggests that transfer cells form as a response to solute flow

Fungal endophytes and a virus confer drought tolerance to Nicotiana benthamiana plants through modulating osmolytes, antioxidant enzymes and expression of host drought-responsive genes

Microbial symbionts increase plant growth and eco-physiological performance under abiotic stress. In this study, we evaluated how the colonization of two fungal endophytes isolated from wild Nicotiana species from areas of drought-prone northern Australia, and a plant virus, yellowtail flower mild mottle virus (genus Tobamovirus), improved water stress tolerance in N. benthamiana plants. Inoculation with both of the two fungal strains used and the virus significantly increased plants tolerance to water stress as manifested by their significant delay in wilting of shoot tips. The water stress tolerance of fungus-inoculated plants was correlated with increases in plant biomass, relative water content, soluble sugar, soluble protein, proline content, increased activities of the antioxidant enzymes catalase, peroxidase and polyphenol oxidase, decreased production of reactive oxygen species, and decreased electrical conductivity. In addition, there was significant upregulation of several genes previously identified as drought induced. The influence of the virus was similar to the fungi in terms of increasing the plant osmolytes, antioxidant enzyme activity and gene expression. Although separate infection of fungi and virus increased plant water stress tolerance responses, their co-infection in plants did not have an additive effect on water stress responses. These findings show that both fungi and viruses reprogram plant responses to water stress in a similar way

Weed recognition using deep learning techniques on class-imbalanced imagery

Context: Most weed species can adversely impact agricultural productivity by competing for nutrients required by high-value crops. Manual weeding is not practical for large cropping areas. Many studies have been undertaken to develop automatic weed management systems for agricultural crops. In this process, one of the major tasks is to recognise the weeds from images. However, weed recognition is a challenging task. It is because weed and crop plants can be similar in colour, texture and shape which can be exacerbated further by the imaging conditions, geographic or weather conditions when the images are recorded. Advanced machine learning techniques can be used to recognise weeds from imagery. Aims: In this paper, we have investigated five state-of-the-art deep neural networks, namely VGG16, ResNet-50, Inception-V3, Inception-ResNet-v2 and MobileNetV2, and evaluated their performance for weed recognition. Methods: We have used several experimental settings and multiple dataset combinations. In particular, we constructed a large weed-crop dataset by combining several smaller datasets, mitigating class imbalance by data augmentation, and using this dataset in benchmarking the deep neural networks. We investigated the use of transfer learning techniques by preserving the pre-trained weights for extracting the features and fine-tuning them using the images of crop and weed datasets. Key results: We found that VGG16 performed better than others on small-scale datasets, while ResNet-50 performed better than other deep networks on the large combined dataset. Conclusions: This research shows that data augmentation and fine tuning techniques improve the performance of deep learning models for classifying crop and weed images. Implications: This research evaluates the performance of several deep learning models and offers directions for using the most appropriate models as well as highlights the need for a large scale benchmark weed dataset

A survey of image-based computational learning techniques for frost detection in plants

Frost damage is one of the major concerns for crop growers as it can impact the growth of the plants and hence, yields. Early detection of frost can help farmers mitigating its impact. In the past, frost detection was a manual or visual process. Image-based techniques are increasingly being used to understand frost development in plants and automatic assessment of damage resulting from frost. This research presents a comprehensive survey of the state-of the-art methods applied to detect and analyse frost stress in plants. We identify three broad computational learning approaches i.e., statistical, traditional machine learning and deep learning, applied to images to detect and analyse frost in plants. We propose a novel taxonomy to classify the existing studies based on several attributes. This taxonomy has been developed to classify the major characteristics of a significant body of published research. In this survey, we profile 80 relevant papers based on the proposed taxonomy. We thoroughly analyse and discuss the techniques used in the various approaches, i.e., data acquisition, data preparation, feature extraction, computational learning, and evaluation. We summarise the current challenges and discuss the opportunities for future research and development in this area including in-field advanced artificial intelligence systems for real-time frost monitoring

Co-infection with three mycoviruses stimulates growth of a Monilinia fructicola isolate on nutrient medium, but does not induce hypervirulence in a natural host

Monilinia fructicola and Monilinia laxa are the most destructive fungal species infecting stone fruit (Prunus species). High-throughput cDNA sequencing of M. laxa and M. fructicola isolates collected from stone fruit orchards revealed that 14% of isolates were infected with one or more of three mycoviruses: Sclerotinia sclerotiorum hypovirus 2 (SsHV2, genus Hypovirus), Fusarium poae virus 1 (FPV1, genus Betapartitivirus), and Botrytis virus F (BVF, genus Mycoflexivirus). Isolate M196 of M. fructicola was co-infected with all three viruses, and this isolate was studied further. Several methods were applied to cure M196 of one or more mycoviruses. Of these treatments, hyphal tip culture either alone or in combination with antibiotic treatment generated isogenic lines free of one or more mycoviruses. When isogenic fungal lines were cultured on nutrient agar medium in vitro, the triple mycovirus-infected parent isolate M196 grew 10% faster than any of the virus-cured isogenic lines. BVF had a slight inhibitory effect on growth, and FPV1 did not influence growth. Surprisingly, after inoculation to fruits of sweet cherry, there were no significance differences in disease progression between isogenic lines, suggesting that these mycoviruses did not influence the virulence of M. fructicola on a natural host

Catharanthus mosaic virus: A potyvirus from a gymnosperm, Welwitschia mirabilis

A virus from a symptomatic plant of the gymnosperm Welwitschia mirabilis Hook. growing as an ornamental plant in a domestic garden in Western Australia was inoculated to a plant of Nicotiana benthamiana where it established a systemic infection. The complete genome sequence of 9636 nucleotides was determined using high-throughput and Sanger sequencing technologies. The genome sequence shared greatest identity (83% nucleotides and 91% amino acids) with available partial sequences of catharanthus mosaic virus, indicating that the new isolate belonged to that taxon. Analysis of the phylogeny of the complete virus sequence placed it in a monotypic group in the genus Potyvirus. This is the first record of a virus from W. mirabilis, the first complete genome sequence of catharanthus mosaic virus determined, and the first record from Australia. This finding illustrates the risk to natural and managed systems posed by the international trade in live plants and propagules, which enables viruses to establish in new regions and infect new hosts