First Report of Fusarium avenaceum Causing Postharvest Decay of European Pear in Mid-Atlantic United States.

Fruit losses due to postharvest decay caused by several fungal species is a major challenge for pear production (Sardella et al. 2016). In December 2021, a European pear (Pyrus communis L.) 'Dawn' with brown, circular, watery, and sunken lesions was observed in cold storage at the USDA Appalachian Fruit Research Station in Kearneysville, West Virginia. Only 1 of 14 'Dawn' pears examined in cold storage had the described disease symptoms. The fruit was surface sterilized, and symptomatic tissue was transferred to potato dextrose agar (PDA) and incubated at 25°C under continuous light. The isolate was hyphal tip purified and propagated on PDA plates at 25°C. The colonies grew an average of 8 mm/day and produced fluffy white aerial mycelium and pigmented rings of golden yellow which then darkened to pink with a dark pink on the reverse. The isolate was also cultured in liquid basal medium with carboxymethyl cellulose (Moura et al. 2020) for 7 days at 25°C to promote macroconidia formation. Macroconidia were slightly falcate with a tapering apical cell, usually 2 to 4 septate, and on average 16.8 µm long by 2.8 µm wide. The isolate was identified as Fusarium spp. based on morphology. Identity of the isolate was confirmed through sequencing of the ITS and TEF1 gene region (Stielow et al. 2015). The ITS and TEF1 sequences were deposited in GenBank (OP007197 and OP007198). BLAST analysis of the ITS amplicon identified multiple Fusarium spp. with 100% identity and 100% query coverage including F. avenaceum KJ562378. BLAST analysis of the TEF1 amplicon showed 99% identity and 99-100% query coverage with F. avenaceum isolates KM189442 and MK512754. Organic Bartlett pears were surface sanitized with a 1% aqueous chlorine solution, rinsed with sterile water and dried in a laminar flow hood. Fruit were then wounded with a sterile nail (4 mm diameter x 4 mm depth) and inoculated with a 4 mm mycelial plug taken from a 7- to 10-day old culture on PDA and wrapped with Parafilm. Plugs taken from sterile PDA were used as a control. Inoculated fruit were stored at 25°C in fruit trays in plastic bins for 7 days. Six fruit composed a replicate, and the experiment was repeated for a total of two replications. Lesions developed within 48 hours and expanded to an average of 28.5 mm by day 7. No lesions were observed on control fruit. Symptoms observed on inoculated pears were the same as the decay observed on the original pear obtained from cold storage. Fungal colonies isolated from the lesions and cultured on PDA morphologically resembled the original isolate from the infected pear. In 2014, F. avenaceum was first reported in the United States to cause post-harvest decay of apples in Pennsylvania (Kou et al. 2014). In the Netherlands, F. avenaceum has been reported to cause postharvest decay of 'Conference' pears but was observed at low frequencies (1-5%) in packing-house surveys (Wenneker et al. 2016). Fusarium spp. was also recently found on European pears in Southern Oregon (KC and Rasmussen 2020). F. avenaceum can produce mycotoxins which is a concern for fruit processing (Munkvold et al. 2021). Monitoring for this pathogen to prevent losses and mycotoxin contamination of processed fruit products will be import for consumer safety. To our knowledge, this is the first report of F. avenaceum causing postharvest decay of European pear in the Mid-Atlantic region of the United States.

Viral Reservoir Capacity of Wild Prunus Alternative Hosts of Plum Pox Virus Through Multiple Cycles of Transmission and Dormancy.

Plum pox virus (PPV) is a significant pathogen of Prunus worldwide and is known for having a broad experimental host range. Many of these hosts represent epidemiological risks as potential wild viral reservoirs. A comparative study of the PPV reservoir capacity of three commonly found native North American species, western choke cherry (Prunus virginiana var. demissa), black cherry (Prunus serotina), and American plum (Prunus americana) was conducted. Pennsylvania isolates of PPV-D were transmitted from the original host peach (Prunus persica cv. GF305) to all three species. Viral accumulation and transmission rates to alternative hosts and peach were monitored over the course of five vegetative growth and cold induced dormancy (CID) cycles. The three alternative host species demonstrated differences in their ability to maintain PPV-D and the likelihood of transmission to additional alternative hosts or back transmission to peach. Western choke cherry had low (5.8%) initial infection levels, PPV-D was not transmissible to additional western choke cherry, and transmission of PPV-D from western choke cherry to peach was only possible before the first CID cycle. Black cherry had intermediate initial infection levels (26.6%) but did not maintain high infection levels after repeated CID cycles. Conversely, American plum had a high level (50%) of initial infection that was not significantly different from initial infection in peach (72.2%) and maintained moderate levels (15 to 25%) of infection and PPV-D transmission to both American plum and peach through all five cycles of CID. Our results indicate that American plum has the greatest potential to act as a reservoir host for Pennsylvania isolates of PPV-D.

Dynamic changes impact the plum pox virus population structure during leaf and bud development.

Plum pox virus (PPV) is a worldwide threat to stone fruit production. Its woody perennial hosts provide a dynamic environment for virus evolution over multiple growing seasons. To investigate the impact seasonal host development plays in PPV population structure, next generation sequencing of ribosome associated viral genomes, termed translatome, was used to assess PPV variants derived from phloem or whole leaf tissues over a range of plum leaf and bud developmental stages. Results show that translatome PPV variants occur at proportionately higher levels in bud and newly developing leaf tissues that have low infection levels while more mature tissues with high infection levels display proportionately lower numbers of viral variants. Additional variant analysis identified distinct groups based on population frequency as well as sets of phloem and whole tissue specific variants. Combined, these results indicate PPV population dynamics are impacted by the tissue type and developmental stage of their host.

Viral Hacks of the Plant Vasculature: The Role of Phloem Alterations in Systemic Virus Infection.

For plant viruses, the ability to load into the vascular phloem and spread systemically within a host is an essential step in establishing a successful infection. However, access to the vascular phloem is highly regulated, representing a significant obstacle to virus loading, movement, and subsequent unloading into distal uninfected tissues. Recent studies indicate that during virus infection, phloem tissues are a source of significant transcriptional and translational alterations, with the number of virus-induced differentially expressed genes being four- to sixfold greater in phloem tissues than in surrounding nonphloem tissues. In addition, viruses target phloem-specific components as a means to promote their own systemic movement and disrupt host defense processes. Combined, these studies provide evidence that the vascular phloem plays a significant role in the mediation and control of host responses during infection and as such is a site of considerable modulation by the infecting virus. This review outlines the phloem responses and directed reprograming mechanisms that viruses employ to promote their movement through the vasculature.

AtDRO1 is nuclear localized in root tips under native conditions and impacts auxin localization.

DEEPER ROOTING 1 (DRO1) contributes to the downward gravitropic growth trajectory of roots upstream of lateral auxin transport in monocots and dicots. Loss of DRO1 function leads to horizontally oriented lateral roots and altered gravitropic set point angle, while loss of all three DRO family members results in upward, vertical root growth. Here, we attempt to dissect the roles of AtDRO1 by analyzing expression, protein localization, auxin gradient formation, and auxin responsiveness in the atdro1 mutant. Current evidence suggests AtDRO1 is predominantly a membrane-localized protein. Here we show that VENUS-tagged AtDRO1 driven by the native AtDRO1 promoter complemented an atdro1 Arabidopsis mutant and the protein was localized in root tips and detectable in nuclei. atdro1 primary and lateral roots showed impairment in establishing an auxin gradient upon gravistimulation as visualized with DII-VENUS, a sensor for auxin signaling and proxy for relative auxin distribution. Additionally, PIN3 domain localization was not significantly altered upon gravistimulation in atdro1 primary and lateral roots. RNA-sequencing revealed differential expression of known root development-related genes in atdro1 mutants. atdro1 lateral roots were able to respond to exogenous auxin and AtDRO1 gene expression levels in root tips were unaffected by the addition of auxin. Collectively, the data suggest that nuclear localization may be important for AtDRO1 function and suggests a more nuanced role for DRO1 in regulating auxin-mediated changes in lateral branch angle. KEY MESSAGE: DEEPER ROOTING 1 (DRO1) when expressed from its native promoter is predominately localized in Arabidopsis root tips, detectable in nuclei, and impacts auxin gradient formation.

Translatome Profiling of Plum Pox Virus-Infected Leaves in European Plum Reveals Temporal and Spatial Coordination of Defense Responses in Phloem Tissues.

Plum pox virus (PPV) is the causative agent of sharka, a devastating disease of stone fruits including peaches, apricots, and plums. PPV infection levels and associated disease symptoms can vary greatly, depending upon the virus strain, host species, or cultivar as well as developmental age of the infected tissues. For example, peaches often exhibit mild symptoms in leaves and fruit while European plums typically display severe chlorotic rings. Systemic virus spread into all host tissues occurs via the phloem, a process that is poorly understood in perennial plant species that undergo a period of dormancy and must annually renew phloem tissues. Currently, little is known about how phloem tissues respond to virus infection. Here, we used translating ribosome affinity purification followed by RNA sequencing to identify phloem- and nonphloem-specific gene responses to PPV infection during leaf development in European plum (Prunus domestica L.). Results showed that, during secondary leaf morphogenesis (4- and 6-week-old leaves), the phloem had a disproportionate response to PPV infection with two- to sixfold more differentially expressed genes (DEGs) in phloem than nonphloem tissues, despite similar levels of viral transcripts. In contrast, in mature 12-week-old leaves, virus transcript levels dropped significantly in phloem tissues but not in nonphloem tissues. This drop in virus transcripts correlated with an 18-fold drop in phloem-specific DEGs. Furthermore, genes associated with defense responses including RNA silencing were spatially coordinated in response to PPV accumulation and were specifically induced in phloem tissues at 4 to 6 weeks. Combined, these findings highlight the temporal and spatial dynamics of leaf tissue responses to virus infection and reveal the importance of phloem responses within a perennial host.

Identification of phloem-associated translatome alterations during leaf development in Prunus domestica L.

Phloem plays a fundamental role in plants by transporting hormones, nutrients, proteins, RNAs, and carbohydrates essential for plant growth and development. However, the identity of the underlying phloem genes and pathways remain enigmatic especially in agriculturally important perennial crops, in part, due to the technical difficulty of phloem sampling. Here, we used two phloem-specific promoters and a translating ribosome affinity purification (TRAP) strategy to characterize the phloem translatome during leaf development at 2, 4, and 6 weeks post vernalization in plum (Prunus domestica L.). Results provide insight into the changing phloem processes that occur during leaf development. These processes included the early activation of DNA replication genes that are likely involved in phloem cell division during leaf expansion, as well as the upregulation of phloem genes associated with sink to source conversion, induction of defense processes, and signaling for reproduction. Combined these results reveal the dynamics of phloem gene expression during leaf development and establish the TRAP system as a powerful tool for studying phloem-specific functions and responses in trees.

Tobacco mosaic virus infection disproportionately impacts phloem associated translatomes in Arabidopsis thaliana and Nicotiana benthamiana.

In this study we use vascular specific promoters and a translating ribosome affinity purification strategy to identify phloem associated translatome responses to infection by tobacco mosaic virus (TMV) in systemic hosts Arabidopsis thaliana ecotype Shahdara and Nicotiana benthamiana. Results demonstrate that in both hosts the number of translatome gene alterations that occurred in response to infection is at least four fold higher in phloem specific translatomes than in non-phloem translatomes. This finding indicates that phloem functions as a key responsive tissue to TMV infection. In addition, host comparisons of translatome alterations reveal both similarities and differences in phloem responses to infection, representing both conserved virus induced phloem alterations involved in promoting infection and virus spread as well as host specific alterations that reflect differences in symptom responses. Combined these results suggest phloem tissues play a disproportion role in the mediation and control of host responses to virus infection.

Tobacco mosaic virus-directed reprogramming of auxin/indole acetic acid protein transcriptional responses enhances virus phloem loading.

Vascular phloem loading has long been recognized as an essential step in the establishment of a systemic virus infection. In this study, an interaction between the replication protein of tobacco mosaic virus (TMV) and phloem-specific auxin/indole acetic acid (Aux/IAA) transcriptional regulators was found to modulate virus phloem loading in an age-dependent manner. Promoter expression studies show that in mature tissues TMV 126/183-kDa-interacting Aux/IAAs predominantly express and accumulate within the nuclei of phloem companion cells (CCs). Furthermore, CC Aux/IAA nuclear localization is disrupted upon infection with an interacting virus. In situ analysis of virus spread shows that the inability to disrupt Aux/IAA CC nuclear localization correlates with a reduced ability to load into the vascular tissue. Subsequent systemic movement assays also demonstrate that a virus capable of disrupting Aux/IAA localization is significantly more competitive at moving out of older plant tissues than a noninteracting virus. Similarly, CC expression and overaccumulation of a degradation-resistant Aux/IAA-interacting protein was found to inhibit TMV accumulation and phloem loading selectively in flowering plants. Transcriptional expression studies demonstrate a role for Aux/IAA-interacting proteins in the regulation of salicylic and jasmonic acid host defense responses as well as virus-specific movement factors, including pectin methylesterase, that are involved in regulating plasmodesmata size-exclusion limits and promoting virus cell-to-cell movement. Combined, these findings indicate that TMV directs the reprogramming of auxin-regulated gene expression within the vascular phloem of mature tissues as a means to enhance phloem loading and systemic spread.

The impact of phytohormones on virus infection and disease.

Phytohormones play a critical role in nearly every aspect of plant biology, including development and pathogen defense. During virus infection disruption of the plant's normal developmental physiology has often been associated with alterations in phytohormone accumulation and signaling. Only recently has evidence emerged describing mechanistically how viruses modulate phytohormone levels and the impact these modulations have on plant physiology and virus biology. From these studies there is an emerging theme of virus directed manipulation of plant hormone responses to disarm defense responses and reprogram the cellular environment to enhance replication and spread. In this review we examine the impact viruses have on plant hormone systems and the effects of this phytohormone manipulation on virus biology.