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Since the domestication of plants, pathogenic fungi have consistently threatened crop production, evolving genetically to develop increased virulence under various selection pressures. Understanding their evolutionary trends is crucial for predicting and designing control measures against future disease outbreaks. This paper reviews the evolution of fungal pathogens from natural habitats to agricultural settings, focusing on eight significant phytopathogens: Pyricularia oryzae, Botrytis cinerea, Puccinia spp., Fusarium graminearum, F. oxysporum, Blumeria graminis, Zymoseptoria tritici, and Colletotrichum spp. Also, we explore the mechanism used to understand evolutionary trends in these fungi. The studied pathogens have evolved in agroecosystems through either (1) introduction from elsewhere; or (2) local origins involving co-evolution with host plants, host shifts, or genetic variations within existing strains. Genetic variation, generated via sexual recombination and various asexual mechanisms, often drives pathogen evolution. While sexual recombination is rare and mainly occurs at the center of origin of the pathogen, asexual mechanisms such as mutations, parasexual recombination, horizontal gene or chromosome transfer, and chromosomal structural variations are predominant. Farming practices like mono-cropping resistant cultivars and prolonged use of fungicides with the same mode of action can drive the emergence of new pathotypes. Furthermore, host range does not necessarily impact pathogen adaptation and evolution. Although halting pathogen evolution is impractical, its pace can be slowed by managing selective pressures, optimizing farming practices, and enforcing quarantine regulations. The study of pathogen evolution has been transformed by advancements in molecular biology, genomics, and bioinformatics, utilizing methods like next-generation sequencing, comparative genomics, transcriptomics and population genomics. However, continuous research remains essential to monitor how pathogens evolve over time and to develop proactive strategies that mitigate their impact on agriculture.

Abstract Background Rapid-cycling Brassica napus (B. napus-RC) has potential as a rapid trait testing system for canola (B. napus) because its life cycle is completed within 2 months while canola usually takes 4 months, and it is susceptible to the same range of diseases and abiotic stress as canola. However, a rapid trait testing system for canola requires the development of an efficient transformation and tissue culture system for B. napus-RC. Furthermore, effectiveness of this system needs to be demonstrated by showing that a particular trait can be rapidly introduced into B. napus-RC plants. Results An in-vitro regeneration protocol was developed for B. napus-RC using 4-day-old cotyledons as the explant. High regeneration percentages, exceeding 70%, were achieved when 1-naphthaleneacetic acid (0.10 mg/L), 6-benzylaminopurine (1.0 mg/L), gibberellic acid (0.01 mg/L) and the ethylene antagonist silver nitrate (5 mg/L) were included in the regeneration medium. An average transformation efficiency of 16.4% was obtained using Agrobacterium-mediated transformation of B. napus-RC cotyledons using Agrobacterium strain GV3101 harbouring a plasmid with an NPTII (kanamycin-selectable) marker gene and the Arabidopsis thaliana cDNA encoding ACYL-COA-BINDING PROTEIN6 (AtACBP6). Transgenic B. napus-RC overexpressing AtACBP6 displayed better tolerance to freezing/frost than the wild type, with enhanced recovery from cellular membrane damage at both vegetative and flowering stages. AtACBP6-overexpressing B. napus-RC plants also exhibited lower electrolyte leakage and improved recovery following frost treatment, resulting in higher yields than the wild type. Ovules from transgenic AtACBP6 lines were better protected from frost than those of the wild type, while the developing embryos of frost-treated AtACBP6-overexpressing plants showed less freezing injury than the wild type. Conclusions This study demonstrates that B. napus-RC can be successfully regenerated and transformed from cotyledon explants and has the potential to be an effective trait testing platform for canola. Additionally, AtACBP6 shows potential for enhancing cold tolerance in canola however, larger scale studies will be required to further confirm this outcome.

Chili anthracnose has long been a threat to chili production worldwide. Capsicum baccatum ‘PBC80’ has been identified as a source of resistance to anthracnose. Recently, a QTL for ripe fruit resistance from ‘PBC80’-derived RILs was located on chromosome 4 (123 Mb) and contained over 80 defense-related genes. To identify the genes most related to anthracnose resistance, a fine map of the QTL region was developed using single-marker analysis. Nine genes were selected from the new QTL (1.12 Mb) to study their expression after being challenged with Colletotrichum scovillei ‘MJ5’ in two different RIL genotypes (Resistance/Resistance or R/R and Susceptible/Susceptible or S/S) at 0, 6 and 12 h. Of the nine genes, LYM2, CQW23_09597, CLF, NFXL1, and PR-14 were significantly up-regulated, compared to the control, in the R/R genotype. ERF was up-regulated in both chili genotypes. However, the expression was relatively and constantly low in the S/S genotype. Most up-regulated genes reached the highest peak (2.3–4.5 fold) at 6 h, except for ERF, which had the highest peak at 12 h (6.4 fold). The earliest and highest expressed gene was a pathogen receptor, LYM2.

Pyrethrum (Tanacetum cinerariifolium) cultivation in Australia, which accounts for the majority of global production of natural insecticidal pyrethrins, is affected by a persistent yield decline which in part is caused by a complex of pathogens. Globisporangium and Pythium species were isolated from crown and roots of pyrethrum plants showing stunting and brown discoloration of crown tissue, and from soil adjacent to diseased plants from yield-decline-affected sites in Tasmania and Victoria, Australia. Ten known Globisporangium species (Globisporangium attrantheridium, G. erinaceum, G. intermedium, G. irregulare, G. macrosporum, G. recalcitrans, G. rostratifingens, G. sylvaticum, G. terrestris and G. ultimum var. ultimum), two new Globisporangium species (Globisporangium capense sp. nov. and Globisporangium commune sp. nov.) and three Pythium species (Pythium diclinum/lutarium, P. tracheiphilum and P. vanterpoolii) were identified through morphological studies and multigene phylogenetic analyses using ITS and Cox1 sequences. Globisporangium ultimum var. ultimum, G. sylvaticum, G. commune sp. nov. and G. irregulare were most abundant. Globisporangium attrantheridium, G. macrosporum and G. terrestris were reported for the first time in Australia. Seven Globisporangium species were pathogenic on both pyrethrum seeds (in vitro assays) and seedlings (glasshouse bioassays), while two Globisporangium species and three Pythium species only caused significant symptoms on pyrethrum seeds. Globisporangium irregulare and G. ultimum var. ultimum were the most aggressive species, causing pyrethrum seed rot, seedling damping-off and significant plant biomass reduction. This is the first report of Globisporangium and Pythium species causing disease in pyrethrum globally and suggests that oomycete species in the family Pythiaceae may have an important role in the yield decline of pyrethrum in Australia.

Chinese kale (Brassica alboglabra) is commonly grown and consumed throughout Asia and is often treated with chemicals to control pests and diseases. In Thailand, public standards, Good Agricultural Practice (GAP), and International Federation of Organic Agriculture Movement (IFOAM) programs were introduced for the cultivation of Chinese kale with minimum input of chemical treatments. Black spot caused by the fungus Sphaerobolus has been affecting the plants grown under IFOAM standards in Chiang Mai, Thailand, for several years. Strongly adhering glebal spore masses of the fungus on leaf and stem surfaces have adversely affected postharvest management, especially in the quality classification of the product. Both morphological and phylogenetic (combined ITS, mtSSU, and EF 1-α dataset) studies confirmed a novel species, S. cuprophilus. Pathogenicity tests involving inoculation of Chinese kale by non-wound and mulch inoculation bioassays resulted in the production of symptoms of black spot and the re-isolation of S. cuprophilus, indicating that the new fungal species is the causal agent of black spots. Inhibitory effects of antagonistic bacteria and chemical fungicides, both allowed for use in plant cultivation under either IFOAM or GAP standards, indicated that Bacillus amyloliquefaciens strains (PBT2 and YMB7), chlorothalonil (20 and 500 ppm) and thiophanate-methyl (500 and 1500 ppm) were the most effective in controlling the growth of the causal fungus by 83 to 93%. However, copper oxychloride (5 to 20 ppm), a recommended chemical in control of downy mildew of Chinese kale, showed hormetic effects on S. cuprophilus by promoting the growth and sporulation of the fungus. The findings of this study provide vital information regarding the association of S. cuprophilus and Chinese kale and will support decisions to manage fungal diseases of this vegetable.

Globally, tomato is the second most cultivated vegetable crop next to potato, preferentially grown in temperate climates. Processing tomatoes are generally produced in field conditions, in which soilborne pathogens have serious impacts on tomato yield and quality by causing diseases of the tomato root system. Major processing tomato-producing countries have documented soilborne diseases caused by a variety of pathogens including bacteria, fungi, nematodes, and oomycetes, which are of economic importance and may threaten food security. Recent field surveys in the Australian processing tomato industry showed that plant growth and yield were significantly affected by soilborne pathogens, especially Fusarium oxysporum and Pythium species. Globally, different management methods have been used to control diseases such as the use of resistant tomato cultivars, the application of fungicides, and biological control. Among these methods, biocontrol has received increasing attention due to its high efficiency, target-specificity, sustainability and public acceptance. The application of biocontrol is a mix of different strategies, such as applying antagonistic microorganisms to the field, and using the beneficial metabolites synthesized by these microorganisms. This review provides a broad review of the major soilborne fungal/oomycete pathogens of the field processing tomato industry affecting major global producers, the traditional and biological management practices for the control of the pathogens, and the various strategies of the biological control for tomato soilborne diseases. The advantages and disadvantages of the management strategies are discussed, and highlighted is the importance of biological control in managing the diseases in field processing tomatoes under the pressure of global climate change.

Abstract Anthracnose of chili (Capsicum spp.) causes major production losses throughout Asia where chili plants are grown. A total of 260 Colletotrichum isolates, associated with necrotic lesions of chili leaves and fruit were collected from chili producing areas of Indonesia, Malaysia, Sri Lanka, Thailand and Taiwan. Colletotrichum truncatum was the most commonly isolated species from infected chili fruit and was readily identified by its falcate spores and abundant setae in the necrotic lesions. The other isolates consisted of straight conidia (cylindrical and fusiform) which were difficult to differentiate to species based on morphological characters. Taxonomic analysis of these straight conidia isolates based on multi-gene phylogenetic analyses (ITS, gapdh, chs-1, act, tub2, his3, ApMat, gs) revealed a further seven known Colletotrichum species, C. endophyticum, C. fructicola, C. karsti, C. plurivorum, C. scovillei, C. siamense and C. tropicale. In addition, three novel species are also described as C. javanense, C. makassarense and C. tainanense, associated with anthracnose of chili fruit in West Java (Indonesia); Makassar, South Sulawesi (Indonesia); and Tainan (Taiwan), respectively. Colletotrichum siamense is reported for the first time causing anthracnose of Capsicum annuum in Indonesia and Sri Lanka. This is also the first report of C. fructicola causing anthracnose of chili in Taiwan and Thailand and C. plurivorum in Malaysia and Thailand. Of the species with straight conidia, C. scovillei (acutatum complex), was the most prevalent throughout the surveyed countries, except for Sri Lanka from where this species was not isolated. Colletotrichum siamense (gloeosporioides complex) was also common in Indonesia, Sri Lanka and Thailand. Pathogenicity tests on chili fruit showed that C. javanense and C. scovillei were highly aggressive, especially when inoculated on non-wounded fruit, compared to all other species. The existence of new, highly aggressive exotic species, such as C. javanense, poses a biosecurity risk to production in countries which do not have adequate quarantine regulations to restrict the entry of exotic pathogens.

The early detection of pathogen infections in plants has become an important aspect of integrated disease management. Although previous research demonstrated the idea of applying digital technologies to monitor and predict plant health status, there is no effective system for detecting pathogen infection before symptomatology appears. This paper presents the use of a low-cost and portable electronic nose coupled with machine learning (ML) models for early disease detection. Several artificial neural network models were developed to predict plant physiological data and classify processing tomato plants and soil samples according to different levels of pathogen inoculum by using e-nose outputs as inputs, plant physiological data, and the level of infection as targets. Results showed that the pattern recognition models based on different infection levels had an overall accuracy of 94.4–96.8% for tomato plants and between 94.81% and 96.22% for soil samples. For the prediction of plant physiological parameters (photosynthesis, stomatal conductance, and transpiration) using regression models or tomato plants, the overall correlation coefficient was 0.97–0.99, with very significant slope values in the range 0.97–1. The performance of all models shows no signs of under or overfitting. It is hence proven accurate and valid to use the electronic nose coupled with ML modeling for effective early disease detection of processing tomatoes and could also be further implemented to monitor other abiotic and biotic stressors.

Species of Paramyrothecium that are reported as plant pathogens and cause leaf spot or leaf blight have been reported on many commercial crops worldwide. In 2019, during a survey of fungi causing leaf spots on plants in Chiang Mai and Mae Hong Son provinces, northern Thailand, 16 isolates from 14 host species across nine plant families were collected. A new species Paramyrothecium vignicola sp. nov. was identified based on morphology and concatenated (ITS, cmdA, rpb2, and tub2) phylogeny. Further, P. breviseta and P. foliicola represented novel geographic records to Thailand, while P. eichhorniae represented a novel host record (Psophocarpus sp., Centrosema sp., Aristolochia sp.). These species were confirmed to be the causal agents of the leaf spot disease through pathogenicity assay. Furthermore, cross pathogenicity tests on Coffea arabica L., Commelina benghalensis L., Glycine max (L.) Merr., and Dieffenbachia seguine (Jacq.) Schott revealed multiple host ranges for these pathogens. Further research is required into the host–pathogen relationship of Paramyrothecium species that cause leaf spot and their management. Biotic and abiotic stresses caused by climate change may affect plant health and disease susceptibility. Hence, proper identification and monitoring of fungal communities in the environment are important to understand emerging diseases and for implementation of disease management strategies.

Colletotrichum spp. are important pathogens of citrus that cause dieback of branches and postharvest disease. Globally, several species of Colletotrichum have been identified as causing anthracnose of citrus. One hundred and sixty-eight Colletotrichum isolates were collected from anthracnose symptoms on citrus stems, leaves, and fruit from Victoria, New South Wales, and Queensland, and from State herbaria in Australia. Colletotrichum australianum sp. nov., C. fructicola, C. gloeosporioides, C. karstii, C. siamense, and C. theobromicola were identified using multi-gene phylogenetic analyses based on seven genomic loci (ITS, gapdh, act, tub2, ApMat, gs, and chs-1) in the gloeosporioides complex and five genomic loci (ITS, tub2, act, chs-1, and his3) in the boninense complex, as well as morphological characters. Several isolates pathogenic to chili (Capsicum annuum), previously identified as C. queenslandicum, formed a clade with the citrus isolates described here as C. australianum sp. nov. The spore shape and culture characteristics of the chili and citrus isolates of C. australianum were similar and differed from those of C. queenslandicum. This is the first report of C. theobromicola isolated from citrus and the first detection of C. karstii and C. siamense associated with citrus anthracnose in Australia.

Conditions have been developed for genetic transformation and insertional mutagenesis in Leifsonia xyli subsp. xyli (Lxx), the causal organism of ratoon stunting disease (RSD), one of the most damaging and intractable diseases of sugarcane internationally. Transformation frequencies ranged from 1 to 10 colony forming units (CFU)/μg of plas-mid DNA using Clavibacter/Escherichia coli shuttle vectors pCG188, pDM302, and pDM306 and ranged from 50 to 500 CFU/μg using cosmid cloning vectors pLAFR3 and pLAFR5-km. The transformation/transposition frequency was 0 to 70 CFU/μg of DNA, using suicide vectors pUCD623 and pSUP2021 containing transposable elements Tn4431 and Tn5, respectively. It was necessary to grow Lxx in media containing 0.1% glycine for electroporation and to amplify large plasmids in a dam¯/dcm¯ E. coli strain and purify the DNA by anion exchange. To keep selection pressure at an optimum, the transformants were grown on nitrocellulose filters (0.2-μm pore size) on media containing the appropriate antibiotics. Transposon Tn4431 containing a promoterless lux operon from Vibrio fischeri and a tetra-cycline-resistance gene was introduced on the suicide vector pUCD623. All but 1% of the putative transposon mutants produce light, indicating transposition into functional Lxx genes. Southern blot analysis of these transformants indicates predominantly single transposon insertions at unique sites. The cosmid cloning vector pLAFR5-km was stably maintained in Lxx. The development of a transformation and transposon mutagenesis system opens the way for molecular analysis of pathogenicity determinants in Lxx.

Abstract ColletotrichumMAT1-1-1MAT1-2-1ColletotrichumMAT1-2-1ColletotrichumColletotrichumVerticilliumColletotrichumMAT1-2Colletotrichum

Colletotrichum tanaceti is an emerging foliar fungal pathogen of commercially grown pyrethrum (Tanacetum cinerariifolium). Despite being reported consistently from field surveys in Australia, the molecular basis of pathogenicity of C. tanaceti on pyrethrum is unknown. Herein, the genome of C. tanaceti (isolate BRIP57314) was assembled de novo and annotated using transcriptomic evidence. The inferred putative pathogenicity gene suite of C. tanaceti comprised a large array of genes encoding secreted effectors, proteases, CAZymes and secondary metabolites. Comparative analysis of its putative pathogenicity gene profiles with those of closely related species suggested that C. tanaceti likely has additional hosts to pyrethrum. The genome of C. tanaceti had a high repeat content and repetitive elements were located significantly closer to genes inferred to influence pathogenicity than other genes. These repeats are likely to have accelerated mutational and transposition rates in the genome, resulting in a rapid evolution of certain CAZyme families in this species. The C. tanaceti genome showed strong signals of Repeat Induced Point (RIP) mutation which likely caused its bipartite nature consisting of distinct gene-sparse, repeat and A-T rich regions. Pathogenicity genes within these RIP affected regions were likely to have a higher evolutionary rate than the rest of the genome. This "two-speed" genome phenomenon in certain Colletotrichum spp. was hypothesized to have caused the clustering of species based on the pathogenicity genes, to deviate from taxonomic relationships. The large repertoire of pathogenicity factors that potentially evolve rapidly due to the plasticity of the genome, indicated that C. tanaceti has a high evolutionary potential. Therefore, C. tanaceti poses a high-risk to the pyrethrum industry. Knowledge of the evolution and diversity of the putative pathogenicity genes will facilitate future research in disease management of C. tanaceti and other Colletotrichum spp.