สนทนาอาจเกิดปนเปื้อนเมล็ด โดย phytopathogens systemicallyผ่าน vasculature โรงงาน ผ่าน pistils ดอกไม้ หรือผ่านเจาะตรงผนังรังไข่ (1) สำหรับ phytobacteria มากที่สุดคือทางเดินทั่วไปของทำลายเมล็ด pericarp ของรังไข่ ตัวอย่าง แบคทีเรียระดับผ่าน stomata บนฝักของถั่วทั่วไป (เขียว vulgaris L.) และถั่ว (Pisumsativum L.) นำไปสู่การทำลายเมล็ด โดยอ้อยกำแพงแสนpv phaseoli และ Pseudomonas syringae pv pisi ตามลำดับ(33,44) มีรายงานสังเกตที่คล้ายกันสำหรับ Clavibacter michiganensismichiganensis ถั่วในมะเขือเทศ (Lycopersicum esculentumL.) flaccumfaciens Curtobacterium flaccumfaciens ถั่วและเมล็ดพืชในเมล็ดถั่ว (เขียว vulgaris L.) ทั่วไป (22,31)Phytobacteria ที่ใส่เมล็ด โดยทางเดินนี้โดยทั่วไปเป็นฝากใน testa แต่พวกเขาอาจยังเป็นเป็นภาษาท้องถิ่นเอนโดสเปิร์มและต่อเนื้อเยื่อ (8,35,36) Pistil บุกรุกเป็นทางเดินอื่น ๆ ของรายงานสำหรับ phytobacteria ทำลายเมล็ดKauffman และ Leben (18) รายงานว่า inoculation ของถั่วเหลืองบลอสซั่มกับ P. syringae ถั่ว glycinea ส่งผลให้รบกวนเมล็ดภายในฝัก symptomless หีบ (2) รายงานว่า เมื่อวอลนัตละอองเกสร เหือดปน x. อัพ juglandis (สาเหตุตัวแทนของโรควอลนัท), ถูกถ่ายโอนไปบลอสซั่มวอลนัตหญิงวอลนัทผลลัพธ์ได้ภายในปนเปื้อนการศึกษา ในทำนองเดียวกัน แวนแดร์หมาป่าและ vander Zouwen (37)reported that cauliflower blossom inoculation with X. campestrispv. campestris-contaminated flies led to internal seed infestation.However, to date the effect of these invasion pathways on thelocation of phytobacteria in seeds has not been studied.Both pistil and pericarp invasion of watermelon seed have beenreported for A. citrulli (7,40). Frankle et al. (7) reported thatwatermelon fruit became infested with A. citrulli when bacteriapenetrated open stomata on the ovary pericarp. This pathway ofingress was limited to immature fruits (2 to 3 weeks postanthesis)as stomata on older fruits became covered with wax that preventedbacterial penetration. Walcott et al. (40) reported thatwatermelon seed infestation by A. citrulli occurred by pistil invasionand resulted in contaminated seeds within asymptomaticfruits. Lessl et al. (24) subsequently reported a positive linearrelationship between A. citrulli blossom inoculum dose andpercentage of infested seed lots. Additionally, using a constitutivegreen fluorescent protein mutant, they observed that A. citrullirapidly colonized the stigmas of female blossoms, penetratedthrough the transmitting tract tissues of the style and entered theovaries by 7 days postinoculation (23). Based on these observations,we hypothesized that the pathway of seed infestation mayinfluence the location of A. citrulli in seeds.Unlike other seedborne phytobacteria, the testa was not a majorsite of A. citrulli accumulation in infested watermelon seeds. A.citrulli was detected in relatively low percentages of testae comparedwith that in embryo and PE layer samples. Additionally,when the testae were removed from pericarp- and pistil-inoculatedseeds, BFB seedling transmission percentage was not significantlyaffected. This indicated that regardless of the invasionpathway, A. citrulli cells that accumulate in the testae may not beepidemiologically significant, especially for seeds that have beenstored for long periods of time. This was unexpected based onreports on other pathosystems where phytobacteria predominantlysurvived in the testae (9,31,35). One possible explanation for thisdiscrepancy is that in watermelon seed testae, A. citrulli cells areexposed to unfavorable conditions that cause populations todecline after harvest. These conditions are influenced by factorslike variation in testae anatomy and phytobacterial sensitivity todesiccation that may vary based on pathogen and plant species(32). For example, testae surface features can provide protectivesites that help bacteria evade stress during storage. Xanthomonascampestris pv. malvacearum was reported to survive in cottonseed fibers that remained attached to the testae after delinting(36). Interestingly, we observed that even with seeds from pistilinoculatedlots, a low percentage of testae was contaminated withA. citrulli. This was unexpected because we hypothesized thatpistil inoculation would deposit the bacterium in the seed embryo.However, Walcott et al. (40) previously observed that fruit pulptissues also became contaminated with A. citrulli by pistil inoculation.Hence, A. citrulli cells present in watermelon pulp couldsubsequently become associated with testae, similar to seeds inpericarp-inoculated fruits.Based on our observations the watermelon seed PE acts as abarrier to A. citrulli invasion and colonization of the embryo viapericarp-inoculation. Irrespective of the pathway of bacterialinvasion, >78% of the PE layer samples was contaminated with A.citrulli. However, a significantly higher percentage of embryosamples was contaminated with A. citrulli in pistil-inoculated(>90%) than in pericarp-inoculated seeds (<9%). Removal oftestae and PE layers significantly reduced BFB seedling transmissionin pericarp-inoculated seeds but not in pistil-inoculatedseeds. Clearly, removal of the PE layer eliminated A. citrulliinoculum from pericarp-inoculated seeds but not from pistilinoculatedseeds, for which 90% of the embryos were contami-nated. As expected, germination was significantly reduced (<51%)for seeds from which the testae and PE layers were removed.Further evidence for the role of the PE layer as a barrier toembryo contamination in pericarp-inoculated seeds was providedby immunofluorescence microscopy. Immunolabeled A. citrulli
cells were observed in both the inner layers of the PE envelope
and the intercellular spaces of parenchyma tissue of the cotyledons/
embryos in pistil-inoculated seeds (Fig. 3C). In contrast, A.
citrulli cells were not observed in the embryos of pericarpinoculated
seeds but on the surfaces of PE layers (Fig. 3D).
We also found that pistil-inoculation circumvented the PE layer
and deposited A. citrulli cells in the embryos of watermelon
seeds. This conclusion was supported by the observation that
significantly higher percentages of embryos were infested with A.
citrulli in pistil-inoculated seeds compared to pericarp-inoculated
seeds. Additionally, removal of PE layers and testae did not affect
BFB transmission for pistil-inoculated seeds, while BFB transmission
was significantly reduced for pericarp-inoculated seeds.
At present, details on the specific pathways of A. citrulli ingress
through watermelon blossom tissues are not available; however,
bacterial deposition might be influenced by the timing of development
of the different seed layers. To date, PE layer development
has not been described in detail for watermelon seed, but in
cucumber seeds the PE layer begins to form by 15 days post-
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