Population genetic analyses of Phytophthora cinnamomi reveals three lineages and movement between natural vegetation and avocado orchards in South Africa

Phytophthora cinnamomi is the causal agent of root rot, canker and dieback of thousands of plant species around the globe. This oomycete not only causes severe economic losses to forestry and agricultural industries, but also threatens the health of various plants in natural ecosystems. In this study, 380 isolates of P. cinnamomi from four avocado production areas and two regions of natural vegetation in South Africa were investigated using 15 microsatellite markers. These populations were found to have a low level of genetic diversity and consisted of isolates from three lineages. Shared genotypes were detected between isolates from avocado orchards and natural vegetation, indicating the movement of isolates between these areas. The population from the Western Cape natural vegetation had the highest genotypic diversity and unique alleles, indicating this could be the point of introduction of P. cinnamomi to South Africa. Index of association analysis suggested that five out of six populations were under linkage disequilibrium suggesting a clonal mode of reproduction whereas genotypes sampled from a recently established avocado orchard in the Western Cape were derived from a randomly recombined population. This study provided novel insights on the genetic diversity and spread of P. cinnamomi in South Africa. It also reported on the predominance of triploidy in natural occurring populations and provided evidence for recombination of P. cinnamomi for the first time. The presence of two dominant genotypes in all avocado production areas in South Africa highlight the importance of considering them in disease management and resistance breeding programmes.

Transcriptome Profiling of ‘Candidatus Liberibacter asiaticus’ in Citrus and Psyllids

‘Candidatus Liberibacter asiaticus’ (Las) is an emergent bacterial pathogen that is associated with the devastating citrus huanglongbing (HLB). Vectored by the Asian citrus psyllid, Las colonizes the phloem tissue of citrus, causing severe damage to infected trees. So far, cultivating pure Las culture in axenic media has not been successful, and dual-transcriptome analyses aiming to profile gene expression in both Las and its hosts have a low coverage of the Las genome because of the low abundance of bacterial RNA in total RNA extracts from infected tissues. Therefore, a lack of understanding of the Las transcriptome remains a significant knowledge gap. Here, we used a bacterial cell enrichment procedure and confidently determined the expression profiles of approximately 84% of the Las genes. Genes that exhibited high expression in citrus include transporters, ferritin, outer membrane porins, specific pilins, and genes involved in phage-related functions, cell wall modification, and stress responses. We also found 106 genes to be differentially expressed in citrus versus Asian citrus psyllids. Genes related to transcription or translation and resilience to host defense response were upregulated in citrus, whereas genes involved in energy generation and the flagella system were expressed to higher levels in psyllids. Finally, we determined the relative expression levels of potential Sec-dependent effectors, which are considered as key virulence factors of Las. This work advances our understanding of HLB biology and offers novel insight into the interactions of Las with its plant host and insect vector.

Titer and Distribution of Candidatus Phytoplasma pruni in Prunus avium

‘Candidatus Phytoplasma pruni’ infection in cherries causes small, misshapen fruit with poor color and taste, rendering the fruit unmarketable. However, this is a disease with a long development cycle and a scattered, non-uniform symptom distribution in the early stages. To better understand the biology as well as the relationship between pathogen titer and disease expression, we carried out seasonal, spatial, and temporal examinations of ‘Ca. P. pruni’ titer and distribution in infected orchard-grown trees. Sequential sampling of heavily infected trees revealed marked seasonal patterns, with differential accumulation in woody stem and leaf tissues, and most notably within fruit in the early stages of development from bloom to pit hardening. Furthermore, mapping phytoplasma distribution and titer in trees at different stages of infection indicated that infection proceeds through a series of stages. Initially, infection spreads basipetally and accumulates in the roots before populating aerial parts of the trees from the trunk upwards, with infection of specific tissues and limbs followed by an increasing phytoplasma titer. Finally, we observed a correlation between phytoplasma titer and symptom severity, with severe symptom onset associated with 3-4 orders of magnitude more phytoplasma than mild symptoms. Cumulatively, these data aid in accurate sampling and management decision making and furthers our understanding of disease development.

The diversity of Passalora fulva Isolates Collected from Tomato Plants in US high tunnels

High tunnels extend the growing season of high value crops, including tomatoes, but the environmental conditions within high tunnels favor the spread of the tomato leaf mold pathogen, Passalora fulva (syn. Cladosporium fulvum). Tomato leaf mold results in defoliation, and if severe, losses in yield. Despite substantial research, little is known regarding the genetic structure and diversity of populations of P. fulva associated with high tunnel tomato production in the United States. From 2016 to 2019, a total of 50 P. fulva isolates were collected from tomato leaf samples in high tunnels in the Northeast and Minnesota. Other Cladosporium species were also isolated from the leaf surfaces. Koch’s postulates were conducted to confirm that P. fulva was the cause of the disease symptoms observed. Race determination experiments revealed that the isolates belonged to either race 0 (six isolates) or race 2 (44 isolates). Polymorphisms were identified within four previously characterized effector genes Avr2, Avr4, Avr4e, and Avr9. The largest number of polymorphisms were observed for Avr2. Both mating type genes, MAT1-1-1 and MAT1-2-1, were present in the isolate collection. For further insights into the pathogen diversity, the 50 isolates were genotyped at 7,514 single-nucleotide polymorphism loci using genotyping-by-sequencing: differentiation by region but not by year was observed. Within the collection of 50 isolates, there were 18 distinct genotypes. Information regarding P. fulva population diversity will enable better management recommendations for growers, as high tunnel production of tomatoes expands.

The C2H2 transcription factor SsZFH1 regulates the size, number and development of apothecia in Sclerotinia sclerotiorum

Sclerotinia sclerotiorum is a notorious phytopathogenic, Ascomycota fungus with a host range of over 600 plant species worldwide. This homothallic, Leotiomycetes species reproduces sexually through a multicellular apothecium which produces and releases ascospores. These ascospores serve as the primary inoculum source for disease initiation in the majority of S. sclerotiorum disease cycles. The regulation of apothecium development for this pathogen and other apothecium-producing fungi remains largely unknown. Here, we report that a C2H2 transcription factor SsZFH1 (zinc finger homologous protein) is necessary for the proper development and maturation of sclerotia and apothecia in S. sclerotiorum and is required for the normal growth rate of hyphae. Furthermore, ΔSszfh1 strains exhibit decreased reactive oxygen species (ROS) accumulation in hyphae, increased melanin deposition and enhanced tolerance to H2O2 in the process of vegetative growth and sclerotia formation. Infection assays on common bean leaves, with thin cuticles, and soybean and tomato leaves, with thick cuticles, suggest that the deletion of Sszfh1 slows the mycelial growth rate, which in turn affect the expansion of leaf lesions. Collectively, our results provide novel insights into the fungal factor mediating maturation of apothecia with additional effects on hyphae and sclerotia development.

In-orchard population dynamics of Erwinia amylovora on apple flower stigmas

Populations of the fire blight pathogen Erwinia amylovora Ea110 on apple flower stigmas were tracked over the course of apple bloom in field studies conducted between 2016 and 2019. In 18 of 23 experiments, flower stigmas inoculated on the 1st day of opening were found to harbor large (106-107 cells / flower) populations of E. amylovora when assessed three to five days post-inoculation. However, populations inoculated on stigmas of flowers that were already open for three days did not reach 106 cells / flower, and populations inoculated on stigmas of flowers that were already open for five days never exceeded 104 cells / flower. During this study, >10-fold increases in E. amylovora stigma populations in a 24-hr time period (termed population surges) were observed on 34.8%, 20.0%, and 4.0% of possible days on 1-day, 3-day, and 5-day open flowers, respectively. Population surges occurred on days with average temperatures as high as 24.5°C and as low as 6.1°C. Experiments incorporating more frequent sampling during days and overnight revealed that many population surges occurred between 10:00 PM and 2:00 AM. A Pearson’s correlation analysis of weather parameters occurring during surge events indicated that population surges were significantly associated with situations where overnight temperatures either increased or remained constant, where wind speed decreased, and where relative humidity increased. This study refines our knowledge of E. amylovora population dynamics and further indicates that E. amylovora is able to infect flowers during exposure to colder field temperatures than previously reported.

Transcriptome analysis and functional validation identify a putative bZIP transcription factor, Fpkapc, that regulates development, stress responses, and virulence in Fusarium pseudograminearum

Fusarium pseudograminearum is a soil-borne, hemibiotrophic phytopathogenic fungus that causes Fusarium crown rot and Fusarium head blight in wheat. The basic leucine zipper proteins (bZIPs) are evolutionarily conserved transcription factors that play crucial roles in a range of growth and developmental processes and the responses to biotic and abiotic stresses. However, the roles of bZIP transcription factors remains unknown in F. pseudograminearum. In this study, a bZIP transcription factor Fpkapc was identified to localize to the nucleus in F. pseudograminearum. A mutant strain (Δfpkapc) was constructed to determine the role of Fpkapc in growth and pathogenicity of F. pseudograminearum. Transcriptomic analyses revealed that many genes involved in basic metabolism and oxidation-reduction processes were down-regulated, whereas many genes involved in metal iron binding were up-regulated in the Δfpkapc strain, compared with the wild type. Correspondingly, the mutant had severe growth defects and displayed abnormal hyphal tips. Conidiation in the Fpkapc mutant was reduced, with more conidia in smaller size and fewer septa than in the wild type. Also, relative to WT, the Δfpkapc strain showed greater replaced by increased tolerance to ion stress, but decreased tolerance to H2O2. The mutant caused smaller disease lesions on wheat and barley plants, but the significantly increased TRI genes expression, compared with the wild type. In summary, Fpkapc plays multiple roles in governing growth, development, stress responses, and virulence in F. pseudograminearum.

Phylogenetic Diversity and Mycotoxin Potential of Emergent Phytopathogens within the Fusarium tricinctum Species Complex

Recent studies on multiple continents indicate members of the Fusarium tricinctum species complex (FTSC) are emerging as prevalent pathogens of small-grain cereals, pulses, and other economically important crops. These understudied fusaria produce structurally diverse mycotoxins, among which enniatins (ENNs) and moniliformin (MON) are the most frequent and of greatest concern to food and feed safety. Herein a large survey of fusaria in the Fusarium Research Center and Agricultural Research Service culture collections was undertaken to assess species diversity and mycotoxin potential within the FTSC. A 151-strain collection originating from diverse hosts and substrates from different agroclimatic regions throughout the world was selected from 460 FTSC strains to represent the breadth of FTSC phylogenetic diversity. Evolutionary relationships inferred from a 5-locus dataset, using maximum likelihood and parsimony, resolved the 151 strains as 24 phylogenetically distinct species, including nine that are new to science. Of the five genes analyzed, nearly full-length phosphate permease sequences contained the most phylogenetically informative characters, establishing its suitability for species-level phylogenetics within the FTSC. Fifteen of the species produced ENNs, MON, the sphingosine analog 2-amino-14,16- dimethyloctadecan-3-ol (AOD), and the toxic pigment aurofusarin (AUR) on a cracked corn kernel substrate. Interestingly, the five earliest diverging species in the FTSC phylogeny (i.e., F. iranicum, F. flocciferum, F. torulosum, Fusarium spp. FTSC 8 and 24) failed to produce AOD and MON, but synthesized ENNs and/or AUR. Moreover, our reassessment of nine published phylogenetic studies on the FTSC identified 11 additional novel taxa, suggesting this complex comprises at least 36 species.

Exogenous melatonin improves pear resistance to Botryosphaeria dothidea by increasing autophagic activity and sugar/organic acid levels

Pear is a perennial deciduous fruit tree of the Rosaceae Pyrus genus, and is one of the main fruit trees worldwide. The pathogen Botryosphaeria dothidea infects pear trees and causes pear ring rot disease. According to our research, exogenous melatonin application enhanced resistance to B. dothidea in pear fruit. Melatonin treatment of pears significantly reduced the diameter of disease spots and enhanced the endogenous melatonin content under B. dothidea inoculation. Compared with H2O treatment, melatonin treatment suppressed the increase in ROS and activated ROS-scavenging enzymes. Treatment with exogenous melatonin maintained AsA-GSH at more reductive status. The expression levels of core autophagic genes and autophagosome formation were elevated by melatonin treatment in pear fruit. The silencing of PbrATG5 in Pyrus pyrifolia conferred sensitivity to inoculation, which was only slightly recovered by melatonin treatment. After inoculation with B. dothidea, exogenous melatonin treatment increased the contents of soluble sugars and organic acids in pear fruits compared with H2O treatment. Our results demonstrated that melatonin enhanced resistance to B. dothidea by increasing the autophagic activity and soluble sugar/organic acid accumulation.

Mapping of crown rust (Puccinia coronata f. sp. avenae) resistance gene Pc54 and a novel quantitative trait locus effective against powdery mildew (Blumeria graminis f. sp. avenae) in the oat (Avena sativa) line Pc54

The Pc54 oat line carries the crown rust resistance gene ‘Pc54’ and an unknown gene effective against powdery mildew. In this study two recombinant inbred line populations were developed to identify the genomic locations of the two genes and producing lists of molecular markers with a potential for marker assisted selection. The RILs and parents were phenotyped for crown rust and powdery mildew in a controlled environment. They were also genotyped using the 6K Illumina Infinium iSelect oat SNP chip. Multiple interval mapping placed Pc54 on the linkage group Mrg02 (chromosome 7D) and the novel powdery mildew QTL ‘QPm.18’ on Mrg18 (chromosome 1A) both in the mapping and validating population. A total of nine and 31 significant molecular markers were identified linked with the Pc54 gene and QPm.18, respectively. Reactions to crown rust inoculations have justified separate identity of Pc54 from other genes and QTL that have previously been reported on Mrg02 except for ’qPCRFd’. Pm3 is the only powdery mildew resistance gene previously mapped on Mrg18. However, the pm3 differential line, Mostyn was susceptible to the powdery mildew race used in this study suggesting that Pm3 and QPm.18 are different genes. Determining the chromosomal locations of Pc54 and QPm.18 is helpful for better understanding the molecular mechanism of resistance to crown rust and powdery mildew in oats. Furthermore, SNPs and SSRs that are closely linked with the genes could be valuable for developing PCR based molecular markers and facilitating the utilization of these genes in oat breeding programs.