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Banana (Musa acuminate L.) is an important tropical fruit in China. During 2019-2020, a new leaf spot disease was observed on banana (M. acuminate L. AAA Cavendish, cv. Formosana) at two orchards o...

Fusarium mangiferae is a major causal agent of mango malformation disease (MMD) worldwide. Rapid and accurate detection of the causal pathogen is the cornerstone of integrated disease management. In this paper, a real-time fluorescence loop-mediated isothermal amplification assay (RealAmp) was developed for quantitative detection of F. mangiferae in China. The LAMP primer set was designed based on a RAPD marker sequence and positive products were amplified only from F. mangiferae isolates, but not from any other species tested, showing a high specificity of the primer sets. The detection limit was approximately 2.26 × 10−4 ng/μl plasmid DNA when mixed with extracted mango DNA. Quantification of the pathogen DNA of MMD in naturally collected samples was no significant difference compared to classic real-time PCR Additionally, RealAmp assay was visual with an improved closed-tube visual detection system making the assay more convenient in diagnostics.

Fusarium wilt, caused by the soil-borne fungal pathogen Fusarium oxysporum f.sp. cubense tropical race 4 (TR 4), is regarded as a major yield-reducing factor in the banana industry worldwide. Not all the varieties of banana have the same level of resistance to TR 4, and understanding the molecular interactions in banana defense responses is an important tool to reveal the unexplained processes that underlie banana resistance to TR 4. A proteomic approach was used to explore how a susceptible variety (Williams) and a resistant variety (GCTCV-119) respond to TR 4 during infestation. Sixteen proteins spots were subjected to MALDI-TOF-MS. All of the proteins identified were related to metabolism, energy, immunity and defense, and unknown function. In the resistant variety, eight spots were more abundance, and six spots were less abundance relative to control. Of these, a total of five protein spots showed a-fold change in at least two time points. In the susceptible variety, five spots were more abundance and three spots were less abundance compared with the control. Of these, six spots changed significantly after challenge with TR 4. Chitinase, beta-1,3-glucanase and superoxide dismutase were further investigated using quantitative real time polymerase chain reaction (qRT-PCR) to determine whether the observed proteome changes are associated with changes in the mRNA levels or are due to post-transcriptional regulation. In the study, beta-1,3-glucanase and chitinase were found only in pathogen-challenged plants of the resistance variety compared with the control plants at 6, 12, 24, 48 and 72 hpi, but shown by qRT-PCR beta-1,3-glucanase and chitinase were expressed both in the resistance and susceptible variety in the roots of TR 4-infected bananas.

During 2009–2011, a dieback disease of mango (Mangifera indica) has recently emerged on mango trees in Panzhihua City, Sichuan province of China. The disease is characterized by large irregular brown-coloured speckles on the petioles and twigs, vascular necrosis and dry leaves and complete twig mortality. Fusarium species were isolated repeatedly from the infected petioles and twigs. The species was identified as Fusarium decemcellulare Brick based on morphology and sequence analysis of Translation Elongation Factor-1alpha (TEF-1α) gene. Koch's postulates were fulfilled by pathogenicity tests on potted mango seedlings. To our knowledge, this is the first record of dieback on mango caused by F. decemcellulare in China.

Twenty-two isolates of Corynespora cassiicola obtained from cucumber, papaya, eggplant, tomato, bean, Vigna, sesame and Hevea rubber (Hevea brasiliensis) were analysed by morphological features, the differences of the ribosomal DNA internal transcribed spacer (rDNA-ITS) region sequence and the inter simple sequence repeat (ISSR) technique. Variability of morphological features was observed among the isolates. Pathogenicity tests showed that isolates from different hosts attacked Hevea rubber. Sequences of two outgroup taxa, C. proliferata and C. citricola, were downloaded from GenBank. The phylogenetic trees were constructed by using the rDNA-ITS region sequences from 24 Corynespora spp. isolates. In this analysis, the 24 sequences grouped into two clusters (A and B). Cluster A consists of sequences from all isolates of C. cassiicola; whereas cluster B consists of the two outgroup taxa, C. proliferata and C. citricola. However, the ITS region is conservative, and is not fit for studying differences among isolates. A total of 114 DNA fragments was amplified with 16 ISSR primers, among which 102 were polymorphic (89.5%). A dendrogram was created by the unweighted pair-group method with arithmetic averaging (UPGMA) analysis, and 22 isolates grouped into three clusters (C, D and E). Cluster C is composed of all of the Hevea rubber isolates, whereas cluster D is composed of nine isolates: four from papaya, five from cucumber, eggplant, bean, vigna and sesame. Cluster E is composed of two isolates from cucumber and tomato. These analyses showed that the genetic diversity was very rich among the tested isolates. There are no correlations between the morphological characteristics or rDNA-ITS region sequences of the 22 isolates and their host or geographical origin, but there is a link between ISSR clusters and their host origins. ISSR markers appear to be useful for intra-species population study in C. cassiicola.

Corynespora leaf fall (CLF) disease, caused by the fungal pathogen Corynespora cassiicola, is a major disease of rubber tree (Hevea brasiliensis) that affects the production of natural rubber. To study its pathogenesis, we developed a C. cassiicola transformant tagged with green fluorescent protein and studied it using fluorescence and confocal laser scanning microscopy. The transformed isolate retained its pathogenicity and grew similarly to the wild-type. Conidia germinated and produced germ tubes 4 h after inoculation, after which the fungus infected leaves through wounds; extended into veins, cells, and intercellular spaces within 24 h; produced abundant mycelia on the surfaces of diseased leaves within 48 h; and began to form conidiophores and conidia within 72 h. The infection process of C. cassiicola on H. brasiliensis was complete within 72 h. Our findings may be useful for predicting the path of CLF disease and evaluating H. brasiliensis resistance to C. cassiicola via leaf colonization studies.

Aims In this study, a real-time fluorescence loop-mediated isothermal amplification (RealAmp) was developed and evaluated for the rapid and quantitative detection of Fusarium oxysporum f. sp. cubense race 4 (R4) in soil. Methods and Results The LAMP primer set was designed based on previously verified RAPD marker sequences, and the RealAmp assay could specifically detect and distinguish R4 isolates from other related species. The detection sensitivity of the RealAmp assay was approx. 3·82 × 103 copies of plasmid DNA or 103 of spores per gram in artificially infested soil, indicating that the method is highly tolerant to inhibitor substances in soil compared to real-time PCR. Combining previously published TR4-specific detection methods with the newly established R4-specific RealAmp assay, an indirect approach to detect and differentiate ST4 isolates was achieved by comparing the detection results of R4 and TR4 simultaneously. The existence of ST4 isolates in China was subsequently confirmed through the developed approach. Conclusion The developed RealAmp assay has been confirmed to be a simple, rapid and effective method to detect R4 in soil, which facilitates to further identify and distinguish ST4 isolates through the comparative analysis of detection results between TR4 and R4 simultaneously. Significance and Impact of the Study The technique is an alternative quantitative detection method, which will be used for a routine detection service for the soil-borne pathogen in China.

Multiple funding organizations and grants were incorrectly omitted from the Funding Statement. The Funding Statement should read: "The work was supported by the Earmarked Fund for China Agriculture Research System (Project No. CARS-32-04), Keystone Science and Technology Plan of Hainan Province (ZDXM20130044), the National Natural Science Foundation of China (31301628) and the Central Nonprofit Scientific Research Institutes for Basic Scientific Research Special Fund (1630042013021). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

Fusarium oxysporum f. sp. cubense (Foc), the causal agent of Fusarium wilt (Panama disease), is one of the most devastating diseases of banana (Musa spp.). The Foc tropical race 4 (TR4) is currently known as a major concern in global banana production. No effective resistance is known in Musa to Foc, and no effective measures for controlling Foc once banana plants have been infected in place. Early and accurate detection of Foc TR4 is essential to protect banana industry and guide banana planting. A real-time fluorescence loop-mediated isothermal amplification assay (RealAmp) was developed for the rapid and quantitative detection of Foc TR4 in soil. The detection limit of the RealAmp assay was approximately 0.4 pg/µl plasmid DNA when mixed with extracted soil DNA or 10(3) spores/g of artificial infested soil, and no cross-reaction with other relative pathogens were observed. The RealAmp assay for quantifying genomic DNA of TR4 was confirmed by testing both artificially and naturally infested samples. Quantification of the soil-borne pathogen DNA of Foc TR4 in naturally infested samples was no significant difference compared to classic real-time PCR (P>0.05). Additionally, RealAmp assay was visual with an improved closed-tube visual detection system by adding SYBR Green I fluorescent dye to the inside of the lid prior to amplification, which avoided the inhibitory effects of the stain on DNA amplification and makes the assay more convenient in the field and could thus become a simple, rapid and effective technique that has potential as an alternative tool for the detection and monitoring of Foc TR4 in field, which would be a routine DNA-based testing service for the soil-borne pathogen in South China.

Pathogenicity tests and inter simple sequence repeat (ISSR) molecular fingerprinting markers were utilized to analyze 24 Corynespora cassiicola isolates obtained from a lot of Hevea clones grown in most rubber nurseries and a few plantations in China. The C. cassiicola isolates were collected from Hainan and Yunnan provinces, China, from 2006 to 2008. The assay of 24 C. cassiicola isolates on detached leaves of four different Hevea rubber clones (genotypes PR 107, Dafeng 95, RRIM 600, and Reyan 7-33-97) indicated that 23 of the isolates were susceptible to RRIM 600, and were therefore considered race 1 except for CC-023. ISSR analysis grouped 24 C. cassiicola isolates into four clusters (A, B, C, and D). Unweighted pair-group method with arithmetic averaging (UPGMA) analysis based on Nei and Li’s coefficient (calculated from the binary matrix data of 103 DNA fragments generated from 16 ISSR primers) indicated that cluster A included 19 isolates from Hainan and Yunnan (this cluster was further divided into two sub clusters (I, II), sub cluster II contained isolate CC-023); clusters B and C comprised of 1 isolates from Hainan, respectively; while cluster D encompassed 3 isolates from Hainan and Yunnan. Pathogenicity tests and ISSR analysis showed that there was no correlation between race structure, the geographical origin of the pathogen and their ISSR clusters because 23 of the isolates belonging to four distinct clusters were considered race 1 except for isolate CC-023. However, most of the isolates with different pathogenicity levels shared the same clades, and furthermore, the ISSR clusters and cology color had an exact correlation. These results should facilitate the development of rubber clones with enhanced resistance against all genetic clusters of C. cassiicola.

Corynespora leaf fall disease of Hevea brasiliensis caused by Corynespora cassiicola has been a major cause of severely reduced rubber latex production in Asia and Africa. The aim of this study was to determine whether physiological races of C. cassiicola exist in the Hainan and Yunnan provinces, China, and to accelerate and simplify the process of diagnosis. We conducted pathogenicity tests with 17 C. cassiicola isolates on detached leaves of four different Hevea rubber clones (genotypesPR107, Dafeng 95,RRIM600 and Reyan 7-33-97). Pathogenicity tests showed that 16 of the isolates were pathogenic to RRIM 600, which confirms these isolates as C. cassiicola race 1. An initial PCR was performed to amplify a portion of the ITS1-5.8S-ITS2 region using a C. cassiicola species-specific primer set (CCF and CCR-2).DNA fragments of 272 bp in length indicated the presence of C. cassiicola. To improve the sensitivity, a nested PCR assay was developed to obtain DNA fragments of the expected length (152 bp) using diluted (1 : 100) amplified product from the initial PCR as the template and species-specific oligonucleotides CCF and CCR-1 as primers. The lowest concentration of fungal DNA that could be detected by nested PCR was 168 fg, while its concentration in the initial PCR was 16.8 pg. Therefore, the sensitivity of detection was enhanced 100-fold using nested PCR. In addition, the pathogen could be detected from artificially infected Hevea rubber trees 3 days after inoculation. Due to its high sensitivity, specificity, and reliability, nested PCR is useful for the screening and certification of young C. cassiicola-free Hevea rubber plants for distribution to commercial growers.

Light microscopy of the prepenetration process of Phyllosticta musarum, the banana freckle disease causal agent, on banana leaf was conducted. Germination of conidia started within 2–3 h after inoculation (AI) in a film of water at 25°C. More than 50 and 90% of conidia germinated at 12 and 72 h AI at 25°C, respectively. The germination of a conidium usually produced one germ tube, and occasionally two germ tubes, which were borne at the side of the conidium. However, only one germ tube developed into a mature appressorium. Appressoria were dark brown in colour and had different shapes. Most of them were round or slightly lobed. Appressorium formation initiated at 6 h AI, and the appressorial formation rate reached more than 50 and 80% at 24 and 48 h AI, respectively. A reddish-brown necrosis of host cells appeared under the appressoria as they were forming and was seen at 120 h AI. Most appressoria were formed in the grooves between adjacent epidermis cells. The pathogen seems to penetrate directly through the epidermal cuticle layer of the host by forming appressoria and infection pegs.

Corynespora cassiicola has been found for the first time in China on Hevea rubber tree (Hevea brasiliensis in south China during a survey in 2006. Symptoms on leaves included fishbone necrosis and spots. Sequence data from the ribosomal ITS operon placed the isolates within the Corynespora cassiicola complex.