Biosecurity risks to human food supply associated with plant-parasitic nematodes.

Biosecurity in agriculture is essential for preventing the introduction and spread of plant-parasitic nematodes (PPNs) which threaten global food security by reducing crop yields and facilitating disease spread. These risks are exacerbated by increased global trade and climate change, which may alter PPN distribution and activity, increasing their impact on agricultural systems. Addressing these challenges is vital to maintaining the integrity of the food supply chain. This review highlights significant advancements in managing PPN-related biosecurity risks within the food supply chain, particularly considering climate change's evolving influence. It discusses the PPN modes of transmission, factors increasing the risk of infestation, the impact of PPNs on food safety and security, and traditional and emerging approaches for detecting and managing these pests. Literature suggests that implementing advanced biosecurity measures could decrease PPN infestation rates by up to 70%, substantially reducing crop yield losses and bolstering food security. Notably, the adoption of modern detection and management techniques, (molecular diagnostics and integrated pest management) and emerging geospatial surveillance and analysis systems (spectral imaging, change-detection analysis) has shown greater effectiveness than traditional methods. These innovations offer promising avenues for enhancing crop health and securing the food supply chain against environmental shifts. The integration of these strategies is crucial, demonstrating the potential to transform biosecurity practices and sustain agricultural productivity in the face of changing climatic conditions. This analysis emphasizes the importance of adopting advanced measures to protect crop health and ensure food supply chain resilience, providing valuable insights for stakeholders across the agricultural sector.

Cellular insights of beech leaf disease reveal abnormal ectopic cell division of symptomatic interveinal leaf areas.

The beech leaf disease nematode, Litylenchus crenatae subsp. mccannii, is recognized as a newly emergent nematode species that causes beech leaf disease (BLD) in beech trees (Fagus spp.) in North America. Changes of leaf morphology before emergence from the bud induced by BLD can provoke dramatic effects on the leaf architecture and consequently to tree performance and development. The initial symptoms of BLD appear as dark green, interveinal banding patterns of the leaf. Despite the fast progression of this disease, the cellular mechanisms leading to the formation of such aberrant leaf phenotype remains totally unknown. To understand the cellular basis of BLD, we employed several types of microscopy to provide an exhaustive characterization of nematode-infected buds and leaves. Histological sections revealed a dramatic cell change composition of these nematode-infected tissues. Diseased bud scale cells were typically hypertrophied and showed a high variability of size. Moreover, while altered cell division had no influence on leaf organogenesis, induction of cell proliferation on young leaf primordia led to a dramatic change in cell layer architecture. Hyperplasia and hypertrophy of the different leaf cell layers, coupled with an abnormal proliferation of chloroplasts especially in the mesophyll cell layers, resulted in the typical interveinal leaf banding. These discrepancies in leaf cell structure were depicted by an abnormal rate of cellular division of the leaf interveinal areas infected by the nematode, promoting significant increase of cell size and leaf thickness. The formation of symptomatic BLD leaves is therefore orchestrated by distinct cellular processes, to enhance the value of these feeding sites and to improve their nutrition status for the nematode. Our findings thus uncover relevant cellular events and provide a structural framework to understand this important disease.

Reproduction of Meloidogyne arenaria race 2 on flue-cured tobacco with putative resistance derived from Nicotiana repanda.

Chemical controls for root-knot nematodes are increasingly restricted due to environmental and human health concerns. Host resistance to these nematodes is key to flue-cured tobacco production in Virginia. Resistance to Meloidogyne incognita races 1 and 3, and race 1 of M. arenaria is imparted by the gene Rk1, which is widely available in commercial flue-cured tobacco. Rk2 imparts increased resistance to M. javanica when stacked with Rk1 and is becoming commercially available. The efficacy of Rk2 against M. arenaria race 2, which is increasingly prevalent in Virginia, is unclear. Greenhouse trials were conducted in 2017 to determine how potential resistance derived from N. repanda compares to the root-knot nematode resistance afforded by Rk1 and Rk2. Trials were arranged in a completely randomized block design and included an entry with traits derived from N. repanda, a susceptible entry and entries possessing Rk1 and/or Rk2. Data collected after 60 days included percent root galling, egg mass counts, and egg counts. Root galling and reproduction were significantly lower on the entry possessing traits derived from N. repanda relative to other entries, suggesting that the N. repanda species may hold a novel source of root-knot nematode resistance for commercial flue-cured tobacco cultivars.

Reproduction of Meloidogyne arenaria race 2 on Flue-cured tobacco possessing resistance genes Rk1 and/or Rk2.

Resistance to Meloidogyne incognita races 1 and 3 and race 1 of M. arenaria is imparted to flue-cured tobacco by the gene Rk1. Meloidogyne arenaria race 2 is not controlled by Rk1 and has become prevalent in Virginia. A second form of resistance effective against M. javanica, Rk2, is also increasingly available commercially. Greenhouse and field trials including a root-knot susceptible cultivar, cultivars homozygous for Rk1 or Rk2, and cultivars possessing both genes were conducted in 2018 and 2019 to investigate the effect of Rk1 and/or Rk2 on parasitism and reproduction of M. arenaria race 2. Plants were inoculated with 5,000 M. arenaria race 2 eggs in the greenhouse or infested by a native nematode population in the field. Data were collected after 28 days (greenhouse) or every 3 weeks following transplant until 18 weeks in the field and included root galling index, nematodes present in roots, egg mass numbers, and egg counts; reproductive indices were also calculated. We found that the combination of Rk1 and Rk2 provides greater resistance to M. arenaria race 2 than either gene alone. While the effect of either gene alone was inconsistent, we did observe some significant reductions in galling and reproduction associated with each gene relative to the susceptible control.

Cellular and Transcriptional Responses of Resistant and Susceptible Cultivars of Alfalfa to the Root Lesion Nematode, Pratylenchus penetrans.

The root lesion nematode (RLN), Pratylenchus penetrans, is a migratory species that attacks a broad range of crops, including alfalfa. High levels of infection can reduce alfalfa forage yields and lead to decreased cold tolerance. Currently, there are no commercially certified varieties with RLN resistance. Little information on molecular interactions between alfalfa and P. penetrans, that would shed light on mechanisms of alfalfa resistance to RLN, is available. To advance our understanding of the host-pathogen interactions and to gain biological insights into the genetics and genomics of host resistance to RLN, we performed a comprehensive assessment of resistant and susceptible interactions of alfalfa with P. penetrans that included root penetration studies, ultrastructural observations, and global gene expression profiling of host plants and the nematode. Several gene-candidates associated with alfalfa resistance to P. penetrans and nematode parasitism genes encoding nematode effector proteins were identified for potential use in alfalfa breeding programs or development of new nematicides. We propose that preformed or constitutive defenses, such as significant accumulation of tannin-like deposits in root cells of the resistant cultivar, could be a key to nematode resistance, at least for the specific case of alfalfa-P. penetrans interaction.

Identification and characterization of the first pectin methylesterase gene discovered in the root lesion nematode Pratylenchus penetrans.

Similar to other plant-parasitic nematodes, root lesion nematodes possess an array of enzymes that are involved in the degradation of the plant cell wall. Here we report the identification of a gene encoding a cell wall-degrading enzyme, pectin methylesterase PME (EC 3.1.1.11), in the root lesion nematode Pratylenchus penetrans. Both genomic and coding sequences of the gene were cloned for this species, that included the presence of four introns which eliminated a possible contamination from bacteria. Expression of the Pp-pme gene was localized in the esophageal glands of P. penetrans as determined by in situ hybridization. Temporal expression of Pp-pme in planta was validated at early time points of infection. The possible function and activity of the gene were assessed by transient expression of Pp-pme in plants of Nicotiana benthamiana plants via a Potato virus X-based vector. To our knowledge, this is the first report on identification and characterization of a PME gene within the phylum Nematoda.

Identification of candidate effector genes of Pratylenchus penetrans.

Pratylenchus penetrans is one of the most important species of root lesion nematodes (RLNs) because of its detrimental and economic impact in a wide range of crops. Similar to other plant-parasitic nematodes (PPNs), P. penetrans harbours a significant number of secreted proteins that play key roles during parasitism. Here, we combined spatially and temporally resolved next-generation sequencing datasets of P. penetrans to select a list of candidate genes aimed at the identification of a panel of effector genes for this species. We determined the spatial expression of transcripts of 22 candidate effectors within the oesophageal glands of P. penetrans by in situ hybridization. These comprised homologues of known effectors of other PPNs with diverse putative functions, as well as novel pioneer effectors specific to RLNs. It is noteworthy that five of the pioneer effectors encode extremely proline-rich proteins. We then combined in situ localization of effectors with available genomic data to identify a non-coding motif enriched in promoter regions of a subset of P. penetrans effectors, and thus a putative hallmark of spatial expression. Expression profiling analyses of a subset of candidate effectors confirmed their expression during plant infection. Our current results provide the most comprehensive panel of effectors found for RLNs. Considering the damage caused by P. penetrans, this information provides valuable data to elucidate the mode of parasitism of this nematode and offers useful suggestions regarding the potential use of P. penetrans-specific target effector genes to control this important pathogen.

Characterization of Lilium longiflorum cv. 'Nellie White' Infection with Root-lesion Nematode Pratylenchus penetrans by Bright-field and Transmission Electron Microscopy.

Lilium longiflorum cv. Nellie White, commonly known as Easter lily, is an important floral crop with an annual wholesale value of over $26 million in the United States. The root-lesion nematode, Pratylenchus penetrans, is a major pest of lily due to the significant root damage it causes. In this study, we investigated the cytological aspects of this plant-nematode interaction using bright-field and transmission electron microscopy. We took advantage of an in vitro culture method to multiply lilies and follow the nematode infection over time. Phenotypic reactions of roots inoculated with P. penetrans were evaluated from 0 to 60 d after nematode infection. Symptom development progressed from initial randomly distributed discrete necrotic areas to advanced necrosis along entire roots of each inoculated plant. A major feature characterizing this susceptible host response to nematode infection was the formation of necrosis, browning, and tissue death involving both root epidermis and cortical cells. Degradation of consecutive cell walls resulted in loss of cell pressure, lack of cytoplasmic integrity, followed by cell death along the intracellular path of the nematode's migration. Pratylenchus penetrans was never seen in the vascular cylinder as the layer of collapsed endodermal cells presumably blocked the progression of nematodes into this area of the roots. This study presents the first detailed cytological characterization of P. penetrans infection of Easter lily plants.

The Pratylenchus penetrans Transcriptome as a Source for the Development of Alternative Control Strategies: Mining for Putative Genes Involved in Parasitism and Evaluation of in planta RNAi.

The root lesion nematode Pratylenchus penetrans is considered one of the most economically important species within the genus. Host range studies have shown that nearly 400 plant species can be parasitized by this species. To obtain insight into the transcriptome of this migratory plant-parasitic nematode, we used Illumina mRNA sequencing analysis of a mixed population, as well as nematode reads detected in infected soybean roots 3 and 7 days after nematode infection. Over 140 million paired end reads were obtained for this species, and de novo assembly resulted in a total of 23,715 transcripts. Homology searches showed significant hit matches to 58% of the total number of transcripts using different protein and EST databases. In general, the transcriptome of P. penetrans follows common features reported for other root lesion nematode species. We also explored the efficacy of RNAi, delivered from the host, as a strategy to control P. penetrans, by targeted knock-down of selected nematode genes. Different comparisons were performed to identify putative nematode genes with a role in parasitism, resulting in the identification of transcripts with similarities to other nematode parasitism genes. Focusing on the predicted nematode secreted proteins found in this transcriptome, we observed specific members to be up-regulated at the early time points of infection. In the present study, we observed an enrichment of predicted secreted proteins along the early time points of parasitism by this species, with a significant number being pioneer candidate genes. A representative set of genes examined using RT-PCR confirms their expression during the host infection. The expression patterns of the different candidate genes raise the possibility that they might be involved in critical steps of P. penetrans parasitism. This analysis sheds light on the transcriptional changes that accompany plant infection by P. penetrans, and will aid in identifying potential gene targets for selection and use to design effective control strategies against root lesion nematodes.

First insights into the genetic diversity of the pinewood nematode in its native area using new polymorphic microsatellite loci.

The pinewood nematode, Bursaphelenchus xylophilus, native to North America, is the causative agent of pine wilt disease and among the most important invasive forest pests in the East-Asian countries, such as Japan and China. Since 1999, it has been found in Europe in the Iberian Peninsula, where it also causes significant damage. In a previous study, 94 pairs of microsatellite primers have been identified in silico in the pinewood nematode genome. In the present study, specific PCR amplifications and polymorphism tests to validate these loci were performed and 17 microsatellite loci that were suitable for routine analysis of B. xylophilus genetic diversity were selected. The polymorphism of these markers was evaluated on nematodes from four field origins and one laboratory collection strain, all originate from the native area. The number of alleles and the expected heterozygosity varied between 2 and 11 and between 0.039 and 0.777, respectively. First insights into the population genetic structure of B. xylophilus were obtained using clustering and multivariate methods on the genotypes obtained from the field samples. The results showed that the pinewood nematode genetic diversity is spatially structured at the scale of the pine tree and probably at larger scales. The role of dispersal by the insect vector versus human activities in shaping this structure is discussed.

Meloidogyne polycephannulata n. sp. (Nematoda: Meloidogynidae), a root-knot nematode parasitizing carrot in Brazil.

Meloidogyne polycephannulata n. sp. is described from specimens collected from an area cultivated with carrot cv. Brasilia, in the city of Rio Paranaíba, in the region of Alto Paranaíba, Minas Gerais State, Brazil. The perineal pattern of the female is circular to ovoid with a high dorsal arch that has widely spaced, coarse annulations. The lateral field may have a deep furrow separating the dorsal and ventral arches. The medial lips are short and wide, whereas the lateral lips are large and triangular. The female stylet is 15-16 µm long with wide knobs, distinctly divided by an indentation in the center. Its tip is slightly curved dorsally. The excretory pore opens 34-65 µm from the anterior end. Females retain eggs and second-stage juveniles in their body cavity, similar to that of the cyst-forming nematodes. Males are 1.3-1.7 mm long and have a high head cap that is rounded and slopes posteriorly. The labial disc is fused to the medial lips. The head region has several irregular annulations that are similar in appearance to the first or second body annules that are likewise irregular, making the head region appear to be extremely large. The stylet of the male is 21-24 µm long; it is slender, and has small, rounded knobs, that are distinctly indented medially and appear heart-shaped. The shaft has several tiny projections throughout its length. Mean second-stage juvenile length is 411.7 µm. The juvenile head cap is elevated, the medial lips are small, and the lateral lips are elongate to triangular-shaped. The head region has several short, incomplete and irregular transverse annulations. The juvenile stylet is 14-23 µm long with small, rounded, and sloping knobs. The thin tail ends with a short hyaline portion that is variable in size (16-26 µm) and with a small, rounded tip. Isozyme profiles of esterases from Meloidogyne javanica show 3 strong bands (SB) at Rm 46, 59, and 66; profiles of M. polycephannulata n. sp. show a SB at Rm 47 and a weak band (WB) at Rm 52; M. petuniae has two SB at Rm 44 and 53; M. phaseoli has a SB at 53, 58, and 64 Rm; M. brasilensis has three SB at Rm 40, 58, and 66 and a WB at Rm 71; M. pisi has a SB at Rm 40, 60, and 64 and two WB at 46 and 50 Rm. Data from sequencing the 18S rDNA region of M. polycephannulata n. sp. confirms that it is different from M. arabicida, M. arenaria, M. ethiopica, M. incognita, M. javanica, M. paranaensis, and M. thailandica. Sequence identity among these eight species ranged between 85 to 93.4%. Meloidogyne polycephannulata n. sp. reproduces very well on carrot and tomato; poorly on pepper; and not at all on cotton, peanut, tobacco, watermelon, and sweet corn.